Expression analysis of KIAA nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer

ABSTRACT

The invention relates to methods for detecting, characterizing, preventing, and treating prostate cancer. KIAA markers are provided, wherein changes in the levels of expression of one or more of the KIAA markers is correlated with the presence of prostate cancer.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional PatentApplication Serial No. 60/253,460, filed Nov. 28, 2000, entitled“Expression Analysis of KIAA Nucleic Acids and Polypeptides Useful inthe Diagnosis and Treatment of Prostate Cancer”. The teachings of theforegoing application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Prostate cancer is the second most common cause of cancer relateddeath and will kill an estimated 37,000 people this year alone. Theprostate gland, which is found exclusively in male mammals, producesseveral regulatory peptides. The prostate gland comprises stromal andepithelium cells, the latter group consisting of columnar secretorycells and basal non-secretory cells. A proliferation of these basalcells, as well as stromal cells gives rise to benign prostatichyperplasia (BPH) which is one common prostate disease. Another commonprostate disease is prostatic adenocarcinoma (CaP), the most common ofthe fatal pathophysiological prostate cancers. Prostatic adenocarcinomainvolves a malignant transformation of epithelial cells in theperipheral region of the prostate gland. Prostatic adenocarcinoma andbenign prostatic hyperplasia are two common prostate diseases which havea high rate of incidence in the aging human male population.Approximately one out of every four males above the age of 55 suffersfrom a prostate disease of some form or another.

[0003] To date, various substances that are synthesized and secreted bynormal, benign and cancerous prostates are used as tumor markers to gainan understanding of the pathogenesis of the various prostate diseasesand in the diagnosis of prostate disease. The three predominant proteinsor peptides secreted by a normal prostate gland are Prostatic AcidPhosphatase (PAP), Prostate Specific Antigen (PSA) and prostatic inhibin(PIP) also known as human seminal plasma inhibin (HSPI). Both PSA andPAP have been studied as tumor markers in the detection of prostatedisease but since both exhibit elevated levels in prostates havingbenign prostatic hyperplasia (BPM) neither marker is specific andtherefore are of limited use.

[0004] Despite the available knowledge, little is known about thegenetic basis underlying prostate cancer disease and theandrogen-regulated genes that may be involved with its progression.Although androgens have been known to play a major role in the biologyof prostate cancer, the full complexity of hormonal regulation has notbeen completely elucidated. Many of these processes involve moleculesassociated with prostate cancer, that remain elusive. In addition, theremay be several known molecules that have not yet been associated withthe pathogenesis of the disease. Accordingly, a need exists foridentifying unknown proteins are associated with prostate disorders andthe nucleic acid sequences encoding them. A need also exists foridentifying known proteins and nucleic acid sequences encoding them,that have not yet been implicated in the pathogenesis of prostatecancer, particularly those that can serve as tumor markers for thediagnosis, prevention, and treatment of prostate disorder.

SUMMARY OF THE INVENTION

[0005] The invention is based, in part, on the discovery of a number ofgenes which are induced or repressed in prostate cancer cells (e.g.,androgen-dependent LNCaP cell-lines). These genes serve as markerssuitable for detection, diagnosis and prognosis of prostate disorders.This invention provides methods and screening assays for the detectionand diagnosis of prostate disorders, such as prostate cancer. Theprimary screening assays detect an alteration in the expression level ofgenes identified as being associated with prostate cancer. Inparticular, this invention provides for the use of KIAA clones, such as,KIAA 18 and KIAA 96, as genetic markers for this detection, diagnosisand prognosis of prostate disorders. A number of human KIAA clones havebeen identified (See Nomura et al, (1994) DNA Res. 1: 27-35; Nomura etal, (1994) DNA Res. 1: 223-229 and Nagase et al, (1995) DNA Res. 2:37-43). KIAA 18 and KIAA 96 have been specially identified as beingregulated by androgen in an LNCaP cell-line. The invention provides foruse of KIAA clones that are up-regulated (increased mRNA and proteinexpression/activated/agonized) or down-regulated (decreased mRNA andprotein expression/suppressed/antagonized) in the presence of androgen.

[0006] Examples of KIAA clones include, but are not limited to KIAAclones that encode, enzymes, such as, kinases, e.g., serine-threoninekinases and tyrosine kinases, phosphatases, transglutaminases, proteins,such as, chaperone proteins, growth factors, oncogenes, transcriptionfactors, antibodies and hormones.

[0007] One examplenary KIAA clone that is upregulated in the presence ofandrogen is KIAA 18. The KIAA 18 clone encodes a protein that is amember of the transglutaminase-like superfamily. Transglutaminasecatalyzes the acyl transfer reaction between peptide-bound glutamineresidues and primary amine groups. With the exception of a few familymembers, such as, plasma factor XIIIa, keratinocyte transglutaminase,and epidermal transglutaminase, the function of transglutaminase (andthe genes that encode for it) remains largely unknown. The present studydemonstrated that KIAA 18 expression was up-regulated in LNCaP cancercells in the presence of androgen. KIAA 18 may be used is associatedwith cell growth regulation including tumor development.

[0008] One examplenary KIAA clone that is down-regulated in the presenceof androgen, is KIAA 18. The KIAA 96 clone encodes a protein thatappears to be a serine-threonine kinase which shares a high homologywith SNF1-related proteins. The expression of KIAA 96 was down-regulatedby androgen in LNCaP prostate cancer cells, suggesting that KIAA 96levels may increase in patients in response to androgen ablationtherapy. In addition, subsequent tissue analysis showed that KIAA 96levels increased with tumor grade. KIAA 96 may be involved in cellularproliferation of recurrent tumors and may be a target for anticancerdrug development.

[0009] In one embodiment, the invention provides a method of assessingwhether a subject is afflicted with prostate cancer, by comparing thelevel of expression of a KIAA marker in a sample from a subject, wherethe marker is selected from the group of KIAA 18 and KIAA 96, to thenormal level of expression of the marker in a control sample, where asignificant difference between the level of expression of the marker inthe sample from the subject and the normal level is an indication thatthe subject is afflicted with prostate cancer. The marker can correspondto a transcribed polynucleotide or portion thereof, where thepolynucleotide includes the marker. In a preferred embodiment, the levelof expression of the marker in the sample differs from the normal levelof expression of the marker in a subject not afflicted with prostatecancer by a factor of at least two, and in an even more preferredembodiment, the expression levels differ by a factor of at least three.In another embodiment, the marker is also not significantly expressed innon-prostate cancer cells.

[0010] In another embodiment, the level of expression of the marker inthe sample of cells obtained from the subject is assessed by detectingthe presence in the sample of a protein corresponding to the marker. Ina particularly preferred embodiment, the presence of the protein isdetected using a reagent which specifically binds with the protein. Inan even more preferred embodiment, the reagent is selected from thegroup of reagents including antibodies, antibody derivatives, and anantibody fragments. In another preferred embodiment, the level ofexpression of the marker in the sample is assessed by detecting thepresence in the sample of a transcribed polynucleotide or portionthereof, where the transcribed polynucleotide includes the marker. In aparticularly preferred embodiment, the transcribed polynucleotide is anmRNA or a cDNA. In another particularly preferred embodiment, the stepof detecting further comprises amplifying the transcribedpolynucleotide.

[0011] In yet another preferred embodiment, the level of expression ofthe marker in the sample is assessed by detecting the presence in thesample of a transcribed polynucleotide which anneals with the marker oranneals with a portion of a polynucleotide under stringent hybridizationconditions, where the polynucleotide includes the marker. In anotherpreferred embodiment, the level of expression in the sample of each ofthe KIAA 18 and KIAA 96 markers independently is compared with thenormal level of expression of each of the KIAA 18 and KIAA 96 markers insamples of the same type obtained form control subjects not afflictedwith prostate cancer, where the level of expression of more than one ofthe markers is significantly altered, relative to the correspondingnormal levels of expression of the markers, is an indication that thesubject is afflicted with prostate cancer.

[0012] In another embodiment, the invention provides a method formonitoring the progression of prostate cancer in a subject, includingdetecting in a subject sample at a first point in time the expression ofmarker, where the marker is selected from the group including themarkers KIAA 18 and KIAA 96, repeating this detection step at asubsequent point in time, and comparing the level of expression detectedin the two detection steps, and monitoring the progression of prostatecancer in the subject using this information. In a preferred embodiment,the marker is selected from the group including the markers KIAA 18 andKIAA 96 and combinations thereof. In another preferred embodiment, themarker corresponds to a transcribed polynucleotide or portion thereof,where the polynucleotide includes the marker. In another preferredembodiment, the sample includes cells obtained from the subject. In aparticularly preferred embodiment, the cells are collected from theprostate gland or blood.

[0013] In another embodiment, the invention provides a method ofassessing the efficacy of a test compound for inhibiting prostate cancerin a subject, including comparing expression of a KIAA 18 marker in afirst sample obtained from the subject which is exposed to or maintainedin the presence of the test compound, to expression of the marker in asecond sample obtained from the subject, where the second sample is notexposed to the test compound, where a significantly lower level ofexpression of the marker in the first sample relative to that in thesecond sample is an indication that the test compound is efficacious forinhibiting prostate cancer in the subject.

[0014] In another embodiment, the invention provides a method ofassessing the efficacy of a test compound for inhibiting prostate cancerin a subject, including comparing expression of a KIAA 96 marker in afirst sample obtained from the subject which is exposed to or maintainedin the presence of the test compound, to expression of the marker in asecond sample obtained from the subject, where the second sample is notexposed to the test compound, where a significantly higher level ofexpression of the marker in the first sample relative to that in thesecond sample is an indication that the test compound is efficacious forinhibiting prostate cancer in the subject. In another embodiment, theinvention provides a method of assessing the efficacy of a therapy forinhibiting prostate cancer in a subject, the method including comparingexpression of a KIAA 18 marker in the first sample obtained from thesubject prior to providing at least a portion of the therapy to thesubject, to expression of the marker in a second sample obtained formthe subject following provision of the portion of the therapy, where asignificantly lower level of expression of the KIAA 18 marker in thesecond sample relative to the first sample is an indication that thetherapy is efficacious for inhibiting prostate cancer in the subject.

[0015] In another embodiment, the invention provides a method ofassessing the efficacy of a therapy for inhibiting prostate cancer in asubject, the method including comparing expression of a KIAA 96 markerin the first sample obtained from the subject prior to providing atleast a portion of the therapy to the subject, to expression of themarker in a second sample obtained form the subject following provisionof the portion of the therapy, where a significantly higher level ofexpression of the KIAA 96 marker in the second sample relative to thefirst sample is an indication that the therapy is efficacious forinhibiting prostate cancer in the subject.

[0016] In another embodiment, the invention provides a method ofassessing the efficacy of a therapy for inhibiting prostate cancer in asubject, the method including comparing expression of a KIAA 18 markerin the first sample obtained from the subject prior to providing atleast a portion of the therapy to the subject, to expression of themarker in a second sample obtained form the subject following provisionof the portion of the therapy, where a significantly reduced level ofexpression of the marker in the second sample relative to the firstsample is an indication that the therapy is efficacious for inhibitingprostate cancer in the subject.

[0017] In another embodiment, the invention provides a method ofassessing the efficacy of a therapy for inhibiting prostate cancer in asubject, the method including comparing expression of a KIAA 96 markerin the first sample obtained from the subject prior to providing atleast a portion of the therapy to the subject, to expression of themarker in a second sample obtained form the subject following provisionof the portion of the therapy, where a significantly enhanced level ofexpression of the marker in the second sample relative to the firstsample is an indication that the therapy is efficacious for inhibitingprostate cancer in the subject.

[0018] In another embodiment, the invention provides a method ofselecting a composition for inhibiting prostate cancer in a subject, themethod including obtaining a sample including cells from a subject,separately maintaining aliquots of the sample in the presence of aplurality of test compositions, comparing expression of a marker in eachof the aliquots, where the marker is up-regulated in the presence ofandrogen, such as KIAA 18, and selecting one of the test compositionswhich induces a lower level of expression of the marker in the aliquotcontaining that test composition, relative to other test compositions.

[0019] In another embodiment, the invention provides a method ofselecting a composition for inhibiting prostate cancer in a subject, themethod including obtaining a sample including cells from a subject,separately maintaining aliquots of the sample in the presence of aplurality of test compositions, comparing expression of a marker in eachof the aliquots, where the marker is down-regulated in the presence ofandrogen, such as KIAA 96, and selecting one of the test compositionswhich induces a higher level of expression of the marker in the aliquotcontaining that test composition, relative to other test compositions.

[0020] In another embodiment, the invention provides a method ofselecting a composition for inhibiting prostate cancer in a subject, themethod including obtaining a sample including cells from a subject,separately maintaining aliquots of the sample in the presence of aplurality of test compositions, comparing expression of a marker in eachof the aliquots, where the marker is selected from the group includingthe markers KIAA 18 and KIAA 96, and selecting one of the testcompositions which induces an enhanced, or reduced level of expressionof the marker, respectively, in the aliquot containing that testcomposition, relative to other test compositions.

[0021] In another embodiment, the invention provides a method ofinhibiting prostate cancer in a subject, including obtaining a sampleincluding cells from a subject, separately maintaining aliquots of thesample in the presence of a plurality of test compositions, comparingexpression of a marker in each of the aliquots, where the marker isselected from the group including the markers KIAA 18 and KIAA 96, andadministering to the subject at least one of the test compositions whichinduces a lower, or higher level of expression of the marker,respectivley, in the aliquot containing that test composition, relativeto other test compositions.

[0022] In another embodiment, the invention provides a method ofassessing the potential of a test compound to trigger prostate cancer ina cell, including maintaining separate aliquots of cells in the presenceand absence of the test compound, and comparing expression of a markerin each of the aliquots, where the marker is selected from the groupincluding the markers KIAA 18 and KIAA 96, where a significantlyenhanced ore reduced level of expression of the marker, respectively, inthe aliquot maintained in the presence of the test compound, relative tothe aliquot maintained in the absence of the test compound, is anindication that the test compound possesses the potential for triggeringprostate cancer in a cell.

[0023] In another embodiment, the invention provides a method ofassessing the potential of a test compound to trigger prostate cancer ina cell, including maintaining separate aliquots of cells in the presenceand absence of the test compound, and comparing expression of a markerin each of the aliquots, where the marker is selected from the groupincluding the markers KIAA 18 and KIAA 96, where a significantlydecreased or increased level of expression of the marker respectively,in the aliquot maintained in the presence of the test compound, relativeto the aliquot maintained in the absence of the test compound, is anindication that the test compound possesses the potential for triggeringprostate cancer in a cell.

[0024] In another embodiment, the invention provides a method oftreating a subject afflicted with prostate cancer in which the marker isup-regulated, by an antisense oligonucleotide complementary to apolynucleotide corresponding to a marker, e.g., KIAA 18. Inhibition of amarker that is up-regulated can also be treated by inhibiting expressionof a gene corresponding to the marker that is up-regulated, e.g., KIAA18.

[0025] In another embodiment, the invention provides a method oftreating a subject afflicted with prostate cancer in which the marker isdown-downregulated by providing to cells of the subject afflicted withprostate cancer a protein corresponding to a marker, e.g., KIAA 96. In apreferred embodiment, the protein is provided to the cells by providinga vector including a polynucleotide encoding the protein to the cells.

[0026] In another embodiment, the invention provides a method ofinhibiting prostate cancer in a subject at risk for developing prostatecancer in which the marker is down-regulated, the method comprisingenhancing expression of a gene corresponding to a marker e.g., KIAA 96.

[0027] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

[0028]FIG. 1A is a bar chart depicting the effect of dihydrotestosterone(DHT) on the growth and PSA production of LNCaP cells plated at 20,000cells/well in a 24-well plate with 1 ml of medium. Cells were treatedwith DHT as shown, and cell growth was determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayon day 3;

[0029]FIG. 1B is a graph depicting the effect of DHT on the growth andPSA production of LNCaP cells plated at 1×10⁶ cells/well in a 175 cm²flask. Cells were treated with or without 10 nM DHT the next day, andwere harvested for RNA preparation and PSA analysis.

[0030]FIG. 2 is a flowchart demonstrating the procedure for RNA samplepreparation, Affymetrix Genechip hybridizations and analysis;

[0031]FIG. 3A is a bar chart depicting the expression profile of PSA inresponse to androgen treatment. The mRNA frequencies are plotted on theY-axis, and the DHT androgen treated and untreated cells for each timepoint plotted on the X-axis;

[0032]FIG. 3B is a bar chart depicting the expression profile of KIAA 18in response to androgen treatment. The mRNA frequencies are plotted onthe Y-axis, and the DHT androgen treated and untreated cells for eachtime point plotted on the X-axis;

[0033]FIG. 3C is a bar chart depicting the expression profile of KIAA 96in response to androgen treatment. The mRNA frequencies are plotted onthe Y-axis, and the DHT androgen treated and untreated cells for eachtime point plotted on the X-axis;

[0034]FIG. 4A is a bar chart demonstrating the quantitative RT-PCRanalysis of PSA. Copy number is plotted on the Y-axis, and the DHTandrogen treated and untreated cells for each time point plotted on theX-axis;

[0035]FIG. 4B is a bar chart demonstrating the quantitative RT-PCRanalysis of KIAA 18. Copy number is plotted on the Y-axis, and the DHTandrogen treated and untreated cells for each time point plotted on theX-axis; and

[0036]FIG. 4C is a bar chart demonstrating the quantitative RT-PCRanalysis of KIAA 96. Copy number is plotted on the Y-axis, and the DHTandrogen treated and untreated cells for each time point plotted on theX-axis.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention relates, in part, to newly discovered correlationbetween the expression of selected markers and the presence of prostatecancer in a subject. The relative levels of expression of these markers,both alone and in combination, have been found to be indicative of apredisposition in the subject to prostate cancer and/or diagnostic ofthe presence or potential presence of prostate cancer in a subject. Theinvention markers KIAA 18 and/or KIAA 96, methods for detecting thepresence or absence of prostate cancer in a sample or subject, andmethods of predicting the incidence of prostate cancer in a sample orsubject. The invention also provides methods by which prostate cancermay be treated, using the markers of the invention.

[0038] The present invention is based, at least in part, on theidentification of genetic markers, KIAA 18 and KIAA 96, which aredifferentially expressed in samples from androgen dependent prostatecancer cells. A panel of 6800 known genes was screened for expression inandrogen dependent prostate cancer cells (see Example 1). Those geneswith statistically significant (p<0.05) differences between the diseasedand normal tissues were identified. This differential expression wasobserved either as a decrease in expression, or an increase inexpression. The expression of these selected genes in androgen dependentprostate cancer cells was assessed by GeneChip analysis, as described inExample 1. KIAA 18 was found to increase in expression in LNCaP prostatecancer cells while KIAA 96 was found to decrease in expression in LNCaPprostate cancer cells.

[0039] As an internal control, the prostate specific antigen (PSA) gene,known in the art to be implicated in prostate cancer, was included toscreen androgen dependent prostate cancer cells. PSA was found to besignificantly increased in expression in androgen dependent prostatecancer cells.

[0040] Accordingly, the present invention pertains to the use of theKIAA 18 and/or KIAA 96 genes (e.g., the DNA or cDNA), the correspondingmRNA transcripts, and the encoded polypeptides as markers for thepresence or risk of development prostate cancer. These markers areuseful to correlate the extent and/or severity of disease. The markerscan also be useful in the treatment of prostate cancer, or in assessingthe efficacy of a treatment for cancer. In addition, the markers canalso be used in screening assays to identify compound or agents thatmodify the expression of the markers and the disease state.

[0041] In one aspect, the invention provides markers whose quantity oractivity is correlated with the presence of prostate cancer. The markersof the invention may be nucleic acid molecules (e.g., DNA, cDNA, or RNA)or polypeptides. These markers are either increased or decreased inquantity or activity in prostate cancer tissue as compared tonon-prostate cancer tissue. For example, the gene designated ‘KIAA 18’(accession number D13643) is increased in expression level in androgendependent prostate cancer cell samples, while the gene designated ‘KIAA96’ (accession number D43636) is decreased in expression level inandrogen dependent prostate cancer cell samples. Both the presence ofincreased or decreased mRNA for these genes, and also increased ordecreased levels of the protein products of these genes serve as markersof prostate cancer. Preferably, increased and decreased levels of themarkers of the invention are increases and decreases of a magnitude thatis statistically significant as compared to appropriate control samples(e.g., samples not affected with prostate cancer). In particularlypreferred embodiments, the marker is increased or decreased relative tocontrol samples by at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-foldor more. Similarly, one skilled in the art will be cognizant of the factthat a preferred detection methodology is one in which the resultingdetection values are above the minimum detection limit of themethodology.

[0042] Measurement of the relative amount of an RNA or protein marker ofthe invention may be by any method known in the art (see, e.g.,Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons:1992). Typical methodologies for RNA detection include RNA extractionfrom a cell or tissue sample, followed by hybridization of a labeledprobe (e.g., a complementary nucleic acid molecule) specific for thetarget RNA to the extracted RNA, and detection of the probe (e.g.,Northern blotting). Typical methodologies for protein detection includeprotein extraction from a cell or tissue sample, followed byhybridization of a labeled probe (e.g., an antibody) specific for thetarget protein to the protein sample, and detection of the probe. Thelabel group can be a radioisotope, a fluorescent compound, an enzyme, oran enzyme co-factor. Detection of specific protein and nucleic acidmolecules may also be assessed by gel electrophoresis, columnchromatography, direct sequencing, or quantitative PCR (in the case ofnucleic acid molecules) among many other techniques well known to thoseskilled in the art.

[0043] In certain embodiments, the genes themselves (e.g., the DNA orcDNA) of KIAA 18 or KIAA 96, may serve as markers for prostate cancer.For example, the absence of nucleic acids corresponding to a gene, suchas by deletion of all or part of the gene, may be correlated withdisease. Similarly, an increase of nucleic acid corresponding to theKIAA 18 or KIAA 96 gene, such as by duplication of the gene, may also becorrelated with disease.

[0044] Detection of the presence or number of copies of all or a part ofa marker gene of the invention may be performed using any method knownin the art. Typically, it is convenient to assess the presence and/orquantity of a DNA or cDNA by Southern analysis, in which total DNA froma cell or tissue sample is extracted, is hybridized with a labeled probe(e.g., a complementary DNA molecule), and the probe is detected. Thelabel group can be a radioisotope, a fluorescent compound, an enzyme, oran enzyme co-factor. Other useful methods of DNA detection and/orquantification include direct sequencing, gel electrophoresis, columnchromatography, and quantitative PCR, as is known by one skilled in theart.

[0045] The invention also encompasses nucleic acid and protein moleculeswhich are structurally different from the molecules described above(e.g., which have a slightly altered nucleic acid or amino acidsequence), but which have the same properties as the molecules above(e.g., encoded amino acid sequence, or which are changed only innonessential amino acid residues). Such molecules include allelicvariants, and are described in greater detail in subsection I.

[0046] In another aspect, the invention provides markers whose quantityor activity is correlated with the severity of prostate cancer. Thesemarkers are either increased or decreased in quantity or activity inprostate cancer tissue in a fashion that is either positively ornegatively correlated with the degree of severity of prostate cancer. Inyet another aspect, the invention provides markers whose quantity oractivity is correlated with a risk in a subject for developing prostatecancer. These markers are either increased or decreased in activity orquantity in direct correlation to the likelihood of the development ofprostate cancer in a subject.

[0047] Each marker may be considered individually, although it is withinthe scope of the invention to provide combinations of two or moremarkers for use in the methods and compositions of the invention toincrease the confidence of the analysis. For example, the markers of afirst panel may each exhibit an increase in quantity or activity inprostate cancer tissue as compared to non-prostate cancer tissue,whereas the markers of a second panel may each exhibit a decrease inquantity or activity in prostate cancer tissue as compared tonon-prostate cancer tissue. Similarly, different panels of markers maybe composed of markers from different tissues, or may representdifferent components of a prostate cancer disease.

[0048] It will also be appreciated by one skilled in the art that themarkers of the invention may conveniently be provided on solid supports.For example, polynucleotides, such as mRNA, may be coupled to an array(e.g., a GeneChip array for hybridization analysis), to a resin (e.g., aresin which can be packed into a column for column chromatography), or amatrix (e.g., a nitrocellulose matrix for northern blot analysis). Theimmobilization of molecules complementary to the marker(s), eithercovalently or noncovalently, permits a discrete analysis of the presenceor activity of each marker in a sample. In an array, for example,polynucleotides complementary to each member of a panel of markers mayindividually be attached to different, known locations on the array. Thearray may be hybridized with, for example, polynucleotides extractedfrom a prostate cell sample from a subject. The hybridization ofpolynucleotides from the sample with the array at any location on thearray can be detected, and thus the presence or quantity of the markerin the sample can be ascertained. In a preferred embodiment, a“GeneChip” array is employed (Affymetrix). Similarly, Western analysesmay be performed on immobilized antibodies specific for differentpolypeptide markers hybridized to a protein sample from a subject.

[0049] It will also be apparent to one skilled in the art that theentire marker protein or nucleic acid molecule need not be conjugated tothe support; a portion of the marker of sufficient length for detectionpurposes (e.g., for hybridization), for example, a portion of the markerwhich is 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100or more nucleotides or amino acids in length may be sufficient fordetection purposes.

[0050] The nucleic acid and protein markers of the invention may beisolated from any tissue or cell of a subject. In a preferredembodiment, the tissue is prostate cells or tissue. However, it will beapparent to one skilled in the art that other tissue samples, includingbodily fluids (e.g., blood, urine, bile, serum, lymph, saliva, mucus andpus) and other tissue samples may also serve as sources from which themarkers of the invention may be isolated, or in which the presence,activity, and/or quantity of the markers of the invention may beassessed. The tissue samples containing one or more of the markersthemselves may be useful in the methods of the invention, and oneskilled in the art will be cognizant of the methods by which suchsamples may be conveniently obtained, stored, and/or preserved.

[0051] Several markers were known prior to the invention to beassociated with prostate cancer, e.g., PSA, PIP and HSP1. These markersare not included with the markers of the invention. However, thesemarkers may be conveniently as controls used in combination with themarkers of the invention in the methods, panels, and kits of theinvention.

[0052] In another aspect, the invention provides methods of making anisolated hybridoma which produces an antibody useful for assessingwhether a patient is afflicted with prostate cancer. In this method, aprotein corresponding to a marker of the invention is isolated (e.g., bypurification from a cell in which it is expressed or by transcriptionand translation of a nucleic acid encoding the protein in vivo or invitro using known methods. A vertebrate, preferably a mammal such as amouse, rat, rabbit, or sheep, is immunized using the isolated protein orprotein fragment. The vertebrate may optionally (and preferably) beimmunized at least one additional time with the isolated protein orprotein fragment, so that the vertebrate exhibits a robust immuneresponse to the protein or protein fragment. Splenocytes are isolatedform the immunized vertebrate and fused with an immortalized cell lineto form hybridomas, using any of a variety of methods well known in theart. Hybridomas formed in this manner are then screened using standardmethods to identify one or more hybridomas which produce an antibodywhich specifically binds with the protein or protein fragment. Theinvention also includes hybridomas made by this method and antibodiesmade using such hybridomas.

[0053] The invention provides methods of assessing prostate cancer, orrisk of developing prostate cancer in a subject. These methods involveisolating a sample from a subject (e.g., a sample containing prostatecancer cells or blood cells), detecting the presence, quantity, and/oractivity of one or more markers of the invention in the sample relativeto a second sample from a subject known not to have prostate cancer. Thelevels of markers in the two samples are compared, and a significantincrease or decrease in one or more markers in the test sample indicatesthe presence or risk of presence prostate cancer in the subject.

[0054] The invention also provides methods of assessing the severity ofprostate cancer in a subject. These methods involve isolating a samplefrom a subject (e.g., a sample containing prostate cancer cells or bloodcells), detecting the presence, quantity, and/or activity of one or moremarkers of the invention in the sample relative to a second sample froma subject known not to have prostate cancer. The levels of markers inthe two samples are compared, and a significant increase or decrease inone or more markers in the test sample is correlated with the degree ofseverity of prostate cancer in the subject.

[0055] The invention also provides methods of treating (e.g., inhibitingprostate cancer in a subject. These methods involve isolating a samplefrom a subject (e.g., a sample containing prostate cancer cells or bloodcells), detecting the presence, quantity, and/or activity of one or moremarkers of the invention in the sample relative to a second sample froma subject known not to have prostate cancer. The levels of markers inthe two samples are compared, and significant increases or decreases inone or more markers in the test sample relative to the control sampleare observed. For markers that are significantly decreased in expressionor activity, the subject may be administered that expressed markerprotein, or may be treated by the introduction of mRNA or DNAcorresponding to the decreased marker (e.g., by gene therapy), tothereby increase the levels of the marker protein in the subject. Formarkers that are significantly increased in expression or activity, thesubject may be administered mRNA or DNA antisense to the increasedmarker (e.g., by gene therapy), or may be administered antibodiesspecific for the marker protein, to thereby decrease the levels of themarker protein in the subject. In this manner, the subject may betreated for prostate cancer.

[0056] The invention also provides methods of preventing the developmentprostate cancer in a subject. These methods involve, for markers thatare significantly decreased in expression or activity, theadministration of that marker protein, or the introduction of mRNA orDNA corresponding to the decreased marker (e.g., by gene therapy), tothereby increase the levels of the marker protein in the subject. Formarkers that are significantly increased in expression or activity, thesubject may be administered mRNA or DNA antisense to the increasedmarker (e.g., by gene therapy), or may be administered antibodiesspecific for the marker protein, to thereby decrease the levels of themarker protein in the subject. In this manner, the development prostatecancer in a subject may be prevented.

[0057] The invention also provides methods of assessing a treatment ortherapy for prostate cancer condition in a subject. These methodsinvolve isolating a sample from a subject (e.g., a sample containingprostate cancer cells or blood cells) suffering from prostate cancer whois undergoing a treatment or therapy, detecting the presence, quantity,and/or activity of one or more markers of the invention in the firstsample relative to a second sample from a subject afflicted prostatecancer who is not undergoing any treatment or therapy for the condition,and also relative to a third sample from a subject unafflicted byprostate cancer. The levels of markers in the three samples arecompared, and significant increases or decreases in one or more markersin the first sample relative to the other samples are observed, andcorrelated with the presence, risk of presence, or severity prostatecancer. By assessing prostate cancer has been lessened or alleviated inthe sample, the ability of the treatment or therapy to treat prostatecancer is also determined.

[0058] The invention also provides methods for diagnosingandrogen-dependent prostate cancer in a subject. The method involvesisolating a sample from a subject (e.g., a sample containing prostatecancer cells or blood cells) who is suffering from prostate cancer,measuring the level of expression of a marker selected from the groupconsisting KIAA 18 and KIAA96 in the presence and absence of androgenand comparing the difference in expression of the markers in thepresence and absence of androgen. The prostate cancer cells are androgendependent if the expression of the marker is altered (e.g., increased ordecreased) in the presence of androgen compared to the absence ofandrogen. The invention also provides methods for determining theefficacy of androgen withdrawal treatment in a subject afflicted withprostate cancer. The method involves detecting in a subject sample at afirst point in time, the expression level of a marker selected from thegroup consisting of KIAA 18 and KIAA 96; and detecting the expressionlevel of a marker at a subsequent point in time occurring after thesubject begins androgen withdrawal treatment. The level of expression ofmarkers detected at the first and second time points is compared. Adecrease in the level of expression indicates that the androgenwithdrawal treatment has decreased efficacy.

[0059] The invention also provides pharmaceutical compositions for thetreatment of prostate cancer. These compositions may include a markerprotein and/or nucleic acid of the invention (e.g., for those markerswhich are decreased in quantity or activity in prostate cancer cellsample versus non-prostate cancer cell sample), and can be formulated asdescribed herein. Alternately, these compositions may include anantibody which specifically binds to a marker protein of the inventionand/or an antisense nucleic acid molecule which is complementary to amarker nucleic acid of the invention (e.g., for those markers which areincreased in quantity or activity in a prostate cancer cell sampleversus non-prostate cancer cell sample), and can be formulated asdescribed herein.

[0060] The invention also provides kits for assessing the presence ofprostate cancer in a sample (e.g., a sample from a subject at risk forprostate cancer), the kit comprising an antibody, wherein the antibodyspecifically binds with a protein corresponding to a marker selectedfrom the group consisting of the markers KIAA 18 and/or KIAA 96.

[0061] The invention further provides kits for assessing the presence ofprostate cancer in a sample from a subject (e.g., a subject at risk forprostate cancer), the kit comprising a nucleic acid probe wherein theprobe specifically binds with a transcribed polynucleotide correspondingto a marker selected from the group consisting of the markers KIAA 18and/or KIAA 96.

[0062] The invention further provides kits for assessing the suitabilityof each of a plurality of compounds for inhibiting prostate cancer in asubject. Such kits include a plurality of compounds to be tested, and areagent for assessing expression of a marker selected from the groupconsisting of one or more of the markers KIAA 18 and/or KIAA 96.

[0063] Modifications to the above-described compositions and methods ofthe invention, according to standard techniques, will be readilyapparent to one skilled in the art and are meant to be encompassed bythe invention.

[0064] To facilitate an understanding of the present invention, a numberof terms and phrases are defined below:

[0065] As used herein, the terms “polynucleotide” and “oligonucleotide”are used interchangeably, and include polymeric forms of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. If present, modifications to the nucleotidestructure may be imparted before or after assembly of the polymer. Thesequence of nucleotides may be interrupted by non-nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component. The term also includes bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this invention that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

[0066] A polynucleotide is composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T);and uracil (U) for guanine when the polynucleotide is RNA. This, theterm “polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can beinputted into databases in a computer having a central processing unitand used for bioinformatics applications such as functional genomics andhomology searching.

[0067] A “gene” includes a polynucleotide containing at least one openreading frame that is capable of encoding a particular polypeptide orprotein after being transcribed and translated. Any of thepolynucleotide sequences described herein may be used to identify largerfragments or full-length coding sequences of the gene with which theyare associated. Methods of isolating larger fragment sequences are knownto those of skill in the art, some of which are described herein.

[0068] A “gene product” includes an amino acid (e.g., peptide orpolypeptide) generated when a gene is transcribed and translated.

[0069] A “probe” when used in the context of polynucleotide manipulationincludes an oligonucleotide that is provided as a reagent to detect atarget present in a sample of interest by hybridizing with the target.Usually, a probe will comprise a label or a means by which a label canbe attached, either before or subsequent to the hybridization reaction.Suitable labels include, but are not limited to radioisotopes,fluorochromes, chemiluminescent compounds, dyes, and proteins, includingenzymes.

[0070] A “primer” includes a short polynucleotide, generally with a free3′—OH group that binds to a target or “template” present in a sample ofinterest by hybridizing with the target, and thereafter promotingpolymerization of a polynucleotide complementary to the target. A“polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are well known inthe art, and are taught, for example, in MacPherson et al., IRL Press atOxford University Press (1991)). All processes of producing replicatecopies of a polynucleotide, such as PCR or gene cloning, arecollectively referred to herein as “replication”. A primer can also beused as a probe in hybridization reactions, such as Southern or Northernblot analyses (see, e.g., Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

[0071] The term “cDNAs” includes complementary DNA, that is mRNAmolecules present in a cell or organism made into cDNA with an enzymesuch as reverse transcriptase. A “cDNA library” includes a collection ofmRNA molecules present in a cell or organism, converted into cDNAmolecules with the enzyme reverse transcriptase, then inserted into“vectors” (other DNA molecules that can continue to replicate afteraddition of foreign DNA). Exemplary vectors for libraries includebacteriophage, viruses that infect bacteria (e.g., lambda phage). Thelibrary can then be probed for the specific cDNA (and thus mRNA) ofinterest.

[0072] A “gene delivery vehicle” includes a molecule that is capable ofinserting one or more polynucleotides into a host cell. Examples of genedelivery vehicles are liposomes, biocompatible polymers, includingnatural polymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, viruses and viral vectors, such as baculovirus,adenovirus, and retrovirus, bacteriophage, cosmid, plasmid, fungalvector and other recombination vehicles typically used in the art whichhave been described for replication and/or expression in a variety ofeukaryotic and prokaryotic hosts. The gene delivery vehicles may be usedfor replication of the inserted polynucleotide, gene therapy as well asfor simply polypeptide and protein expression.

[0073] A “vector” includes a self-replicating nucleic acid molecule thattransfers an inserted polynucleotide into and/or between host cells. Theterm is intended to include vectors that function primarily forinsertion of a nucleic acid molecule into a cell, replication vectorsthat function primarily for the replication of nucleic acid andexpression vectors that function for transcription and/or translation ofthe DNA or RNA. Also intended are vectors that provide more than one ofthe above function.

[0074] A “host cell” is intended to include any individual cell or cellculture which can be or has been a recipient for vectors or for theincorporation of exogenous nucleic acid molecules, polynucleotidesand/or proteins. It also is intended to include progeny of a singlecell. The progeny may not necessarily be completely identical (inmorphology or in genomic or total DNA complement) to the original parentcell due to natural, accidental, or deliberate mutation. The cells maybe prokaryotic or eukaryotic, and include but are not limited tobacterial cells, yeast cells, insect cells, animal cells, and mammaliancells, e.g., murine, rat, simian or human cells.

[0075] The term “genetically modified” includes a cell containing and/orexpressing a foreign gene or nucleic acid sequence which in turnmodifies the genotype or phenotype of the cell or its progeny. This termincludes any addition, deletion, or disruption to a cell's endogenousnucleotides.

[0076] As used herein, “expression” includes the process by whichpolynucleotides are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA, if an appropriateeukaryotic host is selected. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Similarly, aeukaryotic expression vector includes a heterologous or homologouspromoter for RNA polymerase II, a downstream polyadenylation signal, thestart codon AUG, and a termination codon for detachment of the ribosome.Such vectors can be obtained commercially or assembled by the sequencesdescribed in methods well known in the art, for example, the methodsdescribed below for constructing vectors in general.

[0077] “Differentially expressed”, as applied to a gene, includes thedifferential production of mRNA transcribed from a gene or a proteinproduct encoded by the gene. A differentially expressed gene may beoverexpressed or underexpressed as compared to the expression level of anormal or control cell. In one aspect, it includes a differential thatis 2 times, preferably 2.5 times, preferably 3 times, preferably 5 timesor preferably 10 times higher or lower than the expression leveldetected in a control sample. The term “differentially expressed” alsoincludes nucleotide sequences in a cell or tissue which are expressedwhere silent in a control cell or not expressed where expressed in acontrol cell.

[0078] The term “polypeptide” includes a compound of two or more subunitamino acids, amino acid analogs, or peptidomimetics. The subunits may belinked by peptide bonds. In another embodiment, the subunit may belinked by other bonds, e.g., ester, ether, etc. As used herein the term“amino acid” includes either natural and/or unnatural or synthetic aminoacids, including glycine and both the D or L optical isomers, and aminoacid analogs and peptidomimetics. A peptide of three or more amino acidsis commonly referred to as an oligopeptide. Peptide chains of greaterthan three or more amino acids are referred to as a polypeptide or aprotein.

[0079] A “protein kinase” is an enzyme that catalyses proteinphosphorylation by transferring the terminal phosphate from adenosinetriphosphate (ATP) to a side chain of a protein. Protein phosphorylationis a reversible process in which the phosphoprotein is converted back tothe unmodified protein by the action of protein phosphatases. Proteinkinases typically comprise a catalytic domain and a regulatory region.The catalytic and regulatory domains may be on the same subunit (e.g.myosin light chain kinase, and calmodulin dependent protein kinase II)or on separate subunits (e.g. cyclin AMP-dependent protein kinase, andphosphorylase kinase). There are two types of protein kinase, those thatphosphoylate one or more serine/threonine residues on a protein, termedserine threonine kinases, and those that phosphorylate one or moretyrosine residues on a protein, termed tyrosine kinase. There are alsodual specificity kinases that are capable of phosphorylating both theserine/threonine residues and tyrosine residues of a protein. Apreferred example of a protein kinase is the KIAA clone, KIAA 96

[0080] A “transglutaminases” are a family of enzymes which catalyze theformation of simple ε-(γ glutamyl) lysine isopeptide bonds in proteins.The enzymes function by catalyzing an acyl-transfer reaction in whichγ-carboxamide groups on peptide-bound glutamine residues serve as theacyl donors. Although the donor substrate is primarily glutamine, thetransglutaminases differ in their specificity for acceptor substrates.In general, transglutaminases are involved in protein cross-linking.Some examples of transglutaminases include activated Factor XIII,epidermal transglutaminase, and prostate transglutaminase. Preferredexamples of transglutaminase enzymes also include the KIAA clone, KIAA18. For a general discussion of transglutaminases See Folk, (1980) AnnRev Biochem 49:517-531.

[0081] “Hybridization” includes a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

[0082] Hybridization reactions can be performed under conditions ofdifferent “stringency”. The stringency of a hybridization reactionincludes the difficulty with which any two nucleic acid molecules willhybridize to one another. Under stringent conditions, nucleic acidmolecules at least 60%, 65%, 70%, 75% identical to each other remainhybridized to each other, whereas molecules with low percent identitycannot remain hybridized. A preferred, non-limiting example of highlystringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., morepreferably at 60° C., and even more preferably at 6° C.

[0083] When hybridization occurs in an antiparallel configurationbetween two single-stranded polynucleotides, the reaction is called“annealing” and those polynucleotides are described as “complementary”.A double-stranded polynucleotide can be “complementary” or “homologous”to another polynucleotide, if hybridization can occur between one of thestrands of the first polynucleotide and the second. with another) isquantifiable in terms of the proportion of bases in opposing strandsthat are expected to hydrogen bond with each other, according togenerally accepted base-pairing rules.

[0084] An “antibody” includes an immunoglobulin molecule capable ofbinding an epitope present on an antigen. As used herein, the termencompasses not only intact immunoglobulin molecules such as monoclonaland polyclonal antibodies, but also anti-idotypic antibodies, mutants,fragments, fusion proteins, bi-specific antibodies, humanized proteins,and modifications of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity.

[0085] As used herein, the term “prostate cancer” (CaP) refers to theart recognized use of the term which commonly appears in men. The term“prostate cancer” refers to both the appearance of a palpable tumor ofthe prostate, and also to microscopically detectable neoplastic ortransformed cells in the prostate gland. In the latter case, the saidcytologically-detectable prostate cancer may be asymptomatic, in thatneither the patient nor the medical practitioner detects the presence ofthe cancer cells. Cancer cells are generally found in the prostates ofmen who live into their seventies or eighties, however not all of thesemen develop prostate cancer. In the event that prostate cancermetastasizes to additional sites distal to the prostate, the conditionis described as metastatic cancer (MC), to distinguish this conditionfrom organ-confined prostate cancer. CaP fatality results frommetastatic dissemination of prostatic adenocarcinoma cells to distantsites, usually in the axial skeleton.

[0086] As used herein, the term “marker” includes a polynucleotide orpolypeptide molecule which is present or absent, or increased ordecreased in quantity or activity in subjects afflicted with prostatecancer, or in cells involved in prostate cancer. The relative change inquantity or activity of the marker is correlated with the incidence orrisk of incidence of prostate cancer.

[0087] As used herein, the term “panel of markers” includes a group ofmarkers, the quantity or activity of each member of which is correlatedwith the incidence or risk of incidence of prostate cancer. In certainembodiments, a panel of markers may include only those markers which areeither increased or decreased in quantity or activity in subjectsafflicted with or cells involved in prostate cancer. In otherembodiments, a panel of markers may include only those markers presentin a specific tissue type which are correlated with the incidence orrisk of incidence of prostate cancer.

[0088] Various aspects of the invention are described in further detailin the following subsections:

[0089] I. Isolated Nucleic Acid Molecules

[0090] One aspect of the invention pertains to isolated nucleic acidmolecules that either themselves are the genetic markers (e.g., mRNA) ofthe invention, or which encode the polypeptide markers of the invention,or fragments thereof. Another aspect of the invention pertains toisolated nucleic acid fragments sufficient for use as hybridizationprobes to identify the nucleic acid molecules encoding the markers ofthe invention in a sample, as well as nucleotide fragments for use asPCR primers for the amplification or mutation of the nucleic acidmolecules which encode the markers of the invention. As used herein, theterm “nucleic acid molecule” is intended to include DNA molecules (e.g.,cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of theDNA or RNA generated using nucleotide analogs. The nucleic acid moleculecan be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

[0091] The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated marker nucleic acidmolecule of the invention, or nucleic acid molecule encoding apolypeptide marker of the invention, can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

[0092] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of one of the KIAA 18and/or KIAA 96 genes, or a portion thereof, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. Using all or portion of the nucleic acid sequence ofone of the KIAA 18 and/or KIAA 96 genes as a hybridization probe, amarker gene of the invention or a nucleic acid molecule encoding apolypeptide marker of the invention can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook,J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

[0093] A nucleic acid of the invention can be amplified using cDNA, mRNAor alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to marker nucleotidesequences, or nucleotide sequences encoding a marker of the inventioncan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

[0094] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleic acid molecule which is acomplement of the nucleotide sequence of a marker of the invention i.e.,KIAA 18 and/or KIAA 96, or a portion of any of these nucleotidesequences. A nucleic acid molecule which is complementary to such anucleotide sequence is one which is sufficiently complementary to thenucleotide sequence such that it can hybridize to the nucleotidesequence, thereby forming a stable duplex.

[0095] The nucleic acid molecule of the invention, moreover, cancomprise only a portion of the nucleic acid sequence of a marker nucleicacid of the invention, or a gene encoding a marker polypeptide of theinvention, for example, a fragment which can be used as a probe orprimer. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 7 or 15, preferably about 20 or 25 more preferably about 50,75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or moreconsecutive nucleotides of a marker nucleic acid, or a nucleic acidencoding a marker polypeptide of the invention.

[0096] Probes based on the nucleotide sequence of a marker gene or of anucleic acid molecule encoding a marker polypeptide of the invention canbe used to detect transcripts or genomic sequences corresponding to themarker gene(s) and/or marker polypeptide(s) of the invention. Inpreferred embodiments, the probe comprises a label group attachedthereto, e.g., the label group can be a radioisotope, a fluorescentcompound, an enzyme, or an enzyme co-factor. Such probes can be used asa part of a diagnostic test kit for identifying cells or tissue whichmisexpress (e.g., over- or under-express) a marker polypeptide of theinvention, or which have greater or fewer copies of a marker gene of theinvention. For example, a level of a marker polypeptide-encoding nucleicacid in a sample of cells from a subject may be detected, the amount ofmRNA transcript of a gene encoding a marker polypeptide may bedetermined, or the presence of mutations or deletions of a marker geneof the invention may be assessed.

[0097] The invention further encompasses nucleic acid molecules thatdiffer from the nucleic acid sequences of the KIAA 18 and KIAA 96 genesdue to degeneracy of the genetic code and which thus encode the sameproteins as those encoded by the KIAA 18 and/or KIAA 96 genes.

[0098] In addition to the nucleotide sequences of the KIAA 18 and KIAA96 genes it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the proteins encoded by the KIAA 18 and KIAA 96 genes may existwithin a population (e.g., the human population). Such geneticpolymorphism in the KIAA 18 and KIAA 96 genes may exist amongindividuals within a population due to natural allelic variation. Anallele is one of a group of genes which occur alternatively at a givengenetic locus. In addition it will be appreciated that DNA polymorphismsthat affect RNA expression levels can also exist that may affect theoverall expression level of that gene (e.g., by affecting regulation ordegradation). As used herein, the phrase “allelic variant” includes anucleotide sequence which occurs at a given locus or to a polypeptideencoded by the nucleotide sequence. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding a marker polypeptide of the invention.

[0099] Nucleic acid molecules corresponding to natural allelic variantsand homologues of the marker genes, or genes encoding the markerproteins of the invention can be isolated based on their homology to theKIAA 18 and KIAA 96 genes, using the cDNAs disclosed herein, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions.Nucleic acid molecules corresponding to natural allelic variants andhomologues of the marker genes of the invention can further be isolatedby mapping to the same chromosome or locus as the marker genes or genesencoding the marker proteins of the invention.

[0100] In another embodiment, an isolated nucleic acid molecule of theinvention is at least 15, 20, 25, 30, 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100,1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000 or more nucleotidesin length and hybridizes under stringent conditions to a nucleic acidmolecule corresponding to a nucleotide sequence of a marker gene or geneencoding a marker protein of the invention. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 70%, more preferably at least about 80%, evenmore preferably at least about 85% or 90% homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 60° C., and even morepreferably at 65° C. Preferably, an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequenceof one of the genes for KIAA 18 and/or KIAA 96. As used herein, a“naturally-occurring” nucleic acid molecule includes an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

[0101] In addition to naturally-occurring allelic variants of the markergene and gene encoding a marker protein of the invention sequences thatmay exist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into the nucleotide sequencesof the marker genes or genes encoding the marker proteins of theinvention, thereby leading to changes in the amino acid sequence of theencoded proteins, without altering the functional activity of theseproteins. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are conservedamong allelic variants or homologs of a gene (e.g., among homologs of agene from different species) are predicted to be particularly unamenableto alteration.

[0102] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding a marker protein of the invention that containchanges in amino acid residues that are not essential for activity. Suchproteins differ in amino acid sequence from the marker proteins encodedby the KIAA 18 and KIAA 96 genes, yet retain biological activity. In oneembodiment, the protein comprises an amino acid sequence at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to amarker protein of the invention.

[0103] An isolated nucleic acid molecule encoding a protein homologousto a marker protein of the invention can be created by introducing oneor more nucleotide substitutions, additions or deletions into thenucleotide sequence of the gene encoding the marker protein, such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intothe KIAA 18 and KIAA 96 genes of the invention by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of a coding sequence of a gene ofthe invention, such as by saturation mutagenesis, and the resultantmutants can be screened for biological activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

[0104] Another aspect of the invention pertains to isolated nucleic acidmolecules which are antisense to the marker genes and genes encodingmarker proteins of the invention. An “antisense” nucleic acid comprisesa nucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire coding strand of a KIAA 18 and KIAA 96 genes, or to only aportion thereof. In one embodiment, an antisense nucleic acid moleculeis antisense to a “coding region” of the coding strand of a nucleotidesequence of the invention. The term “coding region” includes the regionof the nucleotide sequence comprising codons which are translated intoamino acid. In another embodiment, the antisense nucleic acid moleculeis antisense to a “noncoding region” of the coding strand of anucleotide sequence of the invention. The term “noncoding region”includes 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

[0105] Antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof an mRNA corresponding to a gene of the invention, but more preferablyis an oligonucleotide which is antisense to only a portion of the codingor noncoding region. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0106] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding amarker protein of the invention to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventioninclude direct injection at a tissue site (e.g., in skin).Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

[0107] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0108] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave mRNA transcripts of the genes of the invention tothereby inhibit translation of this mRNA. A ribozyme having specificityfor a marker protein-encoding nucleic acid can be designed based uponthe nucleotide sequence of a gene of the invention, disclosed herein.For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in a markerprotein-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, mRNA transcribedfrom a gene of the invention can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g. Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0109] Alternatively, expression of KIAA 18 and KIAA 96 genes can beinhibited by targeting nucleotide sequences complementary to theregulatory region of these genes (e.g., the promoter and/or enhancers)to form triple helical structures that prevent transcription of the genein target cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y Acad. Sci. 660:27-36; andMaher, L. J. (1992) Bioassays 14(12):807-15.

[0110] In yet another embodiment, the nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & MedicinalChemistry 4 (1): 5-23). As used herein, the terms “peptide nucleicacids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, inwhich the deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe etal. Proc. Natl. Acad. Sci. 93: 14670-675.

[0111] PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, for example,inducing transcription or translation arrest or inhibiting replication.PNAs of the nucleic acid molecules of KIAA 18 and KIAA 96 can also beused in the analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup B.(1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0112] In another embodiment, PNAs can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of the nucleic acid molecules of theinvention can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g.,RNAse H and DNA polymerases), to interact with the DNA portion while thePNA portion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimerascan be performed as described in Hyrup B. (1996) supra and Finn P. J. etal. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chaincan be synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

[0113] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier(see, e.g., PCT Publication No. W089/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent). Finally, theoligonucleotide may be detectably labeled, either such that the label isdetected by the addition of another reagent (e.g., a substrate for anenzymatic label), or is detectable immediately upon hybridization of thenucleotide (e.g., a radioactive label or a fluorescent label (e.g., amolecular beacon, as described in U.S. Pat. No. 5,876,930.

[0114] II. Isolated Proteins and Antibodies One aspect of the inventionpertains to isolated marker proteins, and biologically active portionsthereof, as well as polypeptide fragments suitable for use as immunogensto raise anti-marker protein antibodies. In one embodiment, nativemarker proteins can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, marker proteins are produced byrecombinant DNA techniques. Alternative to recombinant expression, amarker protein or polypeptide can be synthesized chemically usingstandard peptide synthesis techniques.

[0115] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which themarker protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofmarker protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of marker protein having lessthan about 30% (by dry weight) of non-marker protein (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-marker protein, still more preferably less than about 10% ofnon-marker protein, and most preferably less than about 5% non-markerprotein. When the marker protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

[0116] The language “substantially free of chemical precursors or otherchemicals” includes preparations of marker protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of protein having less than about 30% (by dryweight) of chemical precursors or non-protein chemicals, more preferablyless than about 20% chemical precursors or non-protein chemicals, stillmore preferably less than about 10% chemical precursors or non-proteinchemicals, and most preferably less than about 5% chemical precursors ornon-protein chemicals.

[0117] As used herein, a “biologically active portion” of a markerprotein includes a fragment of a marker protein comprising amino acidsequences sufficiently homologous to or derived from the amino acidsequence of the marker protein, which include fewer amino acids than thefull length marker proteins, and exhibit at least one activity of amarker protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the marker protein. Abiologically active portion of a marker protein can be a polypeptidewhich is, for example, 10, 25, 50, 100, 200 or more amino acids inlength. Biologically active portions of a marker protein can be used astargets for developing agents which modulate a marker protein-mediatedactivity.

[0118] In a preferred embodiment, marker protein is encoded by the KIAA18 and KIAA 96 genes. In other embodiments, the marker protein issubstantially homologous to a marker protein encoded by the KIAA 18 andKIAA 96 genes, and retains the functional activity of the markerprotein, yet differs in amino acid sequence due to natural allelicvariation or mutagenesis, as described in detail in subsection I above.Accordingly, in another embodiment, the marker protein is a proteinwhich comprises an amino acid sequence at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino acidsequence encoded by the KIAA 18 and KIAA 96 genes.

[0119] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g. gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0120] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

[0121] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to marker proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0122] The invention also provides chimeric or fusion marker proteins.As used herein, a marker “chimeric protein” or “fusion protein”comprises a marker polypeptide operatively linked to a non-markerpolypeptide. An “marker polypeptide” includes a polypeptide having anamino acid sequence encoded by the KIAA 18 and KIAA 96 genes, whereas a“non-marker polypeptide” includes a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the marker protein, e.g., a protein which is differentfrom marker protein and which is derived from the same or a differentorganism. Within a marker fusion protein the polypeptide can correspondto all or a portion of a marker protein. In a preferred embodiment, amarker fusion protein comprises at least one biologically active portionof a marker protein. Within the fusion protein, the term “operativelylinked” is intended to indicate that the marker polypeptide and thenon-marker polypeptide are fused in-frame to each other. The non-markerpolypeptide can be fused to the N-terminus or C-terminus of the markerpolypeptide.

[0123] For example, in one embodiment, the fusion protein is aGST-marker fusion protein in which the marker sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant marker proteins.

[0124] In another embodiment, the fusion protein is a marker proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofmarker proteins can be increased through use of a heterologous signalsequence. Such signal sequences are well known in the art.

[0125] The marker fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo,as described herein. The marker fusion proteins can be used to affectthe bioavailability of a marker protein substrate. Use of marker fusionproteins may be useful therapeutically for the treatment of disorders(e.g., prostate cancer) caused by, for example, (i) aberrantmodification or mutation of a gene encoding a markerprotein; (ii)mis-regulation of the marker protein-encoding gene; and (iii) aberrantpost-translational modification of a marker protein.

[0126] Moreover, the marker-fusion proteins of the invention can be usedas immunogens to produce anti-marker protein antibodies in a subject, topurify marker protein ligands and in screening assays to identifymolecules which inhibit the interaction of a marker protein with amarker protein substrate.

[0127] Preferably, a marker chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A markerprotein-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the markerprotein.

[0128] A signal sequence can be used to facilitate secretion andisolation of the secreted protein or other proteins of interest. Signalsequences are typically characterized by a core of hydrophobic aminoacids which are generally cleaved from the mature protein duringsecretion in one or more cleavage events. Such signal peptides containprocessing sites that allow cleavage of the signal sequence from themature proteins as they pass through the secretory pathway. Thus, theinvention pertains to the described polypeptides having a signalsequence, as well as to polypeptides from which the signal sequence hasbeen proteolytically cleaved (i.e., the cleavage products). In oneembodiment, a nucleic acid sequence encoding a signal sequence can beoperably linked in an expression vector to a protein of interest, suchas a protein which is ordinarily not secreted or is otherwise difficultto isolate. The signal sequence directs secretion of the protein, suchas from a eukaryotic host into which the expression vector istransformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by art recognized methods. Alternatively, the signal sequence canbe linked to the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

[0129] The present invention also pertains to variants of the markerproteins of the invention which function as either agonists (mimetics)or as antagonists to the marker proteins. Variants of the markerproteins can be generated by mutagenesis, e.g., discrete point mutationor truncation of a marker protein. An agonist of the marker proteins canretain substantially the same, or a subset, of the biological activitiesof the naturally occurring form of a marker protein. An antagonist of amarker protein can inhibit one or more of the activities of thenaturally occurring form of the marker protein by, for example,competitively modulating an activity of a marker protein. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the markerprotein.

[0130] Variants of a marker protein which function as either markerprotein agonists (mimetics) or as marker protein antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of a marker protein for marker protein agonist orantagonist activity. In one embodiment, a variegated library of markerprotein variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of marker protein variants can be produced by, forexample, enzymatically ligating a mixture of synthetic oligonucleotidesinto gene sequences such that a degenerate set of potential markerprotein sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of marker protein sequences therein. Thereare a variety of methods which can be used to produce libraries ofpotential marker protein variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential marker proteinsequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

[0131] In addition, libraries of fragments of a protein coding sequencecorresponding to a marker protein of the invention can be used togenerate a variegated population of marker protein fragments forscreening and subsequent selection of variants of a marker protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a marker protein codingsequence with a nuclease under conditions wherein nicking occurs onlyabout once per molecule, denaturing the double stranded DNA, renaturingthe DNA to form double stranded DNA which can include sense/antisensepairs from different nicked products, removing single stranded portionsfrom reformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the marker protein.

[0132] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. The most widely used techniques, which are amenableto high through-put analysis, for screening large gene librariestypically include cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors, and expressing the combinatorial genes under conditions inwhich detection of a desired activity facilitates isolation of thevector encoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify marker variants (Arkin and Yourvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) ProteinEngineering 6(3):327-331).

[0133] An isolated marker protein, or a portion or fragment thereof, canbe used as an immunogen to generate antibodies that bind marker proteinsusing standard techniques for polyclonal and monoclonal antibodypreparation. A full-length marker protein can be used or, alternatively,the invention provides antigenic peptide fragments of these proteins foruse as immunogens. The antigenic peptide of a marker protein comprisesat least 8 amino acid residues of an amino acid sequence encoded by theKIAA 18 and/or KIAA 96 gene, and encompasses an epitope of a markerprotein such that an antibody raised against the peptide forms aspecific immune complex with the marker protein. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

[0134] Preferred epitopes encompassed by the antigenic peptide areregions of the marker protein that are located on the surface of theprotein, e.g., hydrophilic regions, as well as regions with highantigenicity.

[0135] A marker protein immunogen typically is used to prepareantibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, recombinantly expressed markerprotein or a chemically synthesized marker polypeptide. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic marker protein preparation induces apolyclonal anti-marker protein antibody response.

[0136] Accordingly, another aspect of the invention pertains toanti-marker protein antibodies. The term “antibody” as used hereinincludes immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as a marker protein. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind tomarker proteins. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, includes a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope. A monoclonalantibody composition thus typically displays a single binding affinityfor a particular marker protein with which it immunoreacts.

[0137] Polyclonal anti-marker protein antibodies can be prepared asdescribed above by immunizing a suitable subject with a marker proteinof the invention. The anti-marker protein antibody titer in theimmunized subject can be monitored over time by standard techniques,such as with an enzyme linked immunosorbent assay (ELISA) usingimmobilized marker protein. If desired, the antibody molecules directedagainst marker proteins can be isolated from the mammal (e.g., from theblood, or tumor tissue sample) and further purified by well knowntechniques, such as protein A chromatography, to obtain the IgGfraction. At an appropriate time after immunization, e.g., when theanti-marker protein antibody titers are highest, antibody-producingcells can be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975) Nature 256:495-497)(see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al.(1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad.Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75),the more recent human B cell hybridoma technique (Kozbor et al. (1983)Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985),Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)or trioma techniques.

[0138] The technology for producing monoclonal antibody hybridomas iswell known (see generally R. H. Kenneth, in Monoclonal Antibodies: A NewDimension In Biological Analyses, Plenum Publishing Corp., New York, NewYork (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L.Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortalcell line (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with a marker protein immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds to a marker protein of the invention.

[0139] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-marker protein monoclonal antibody (see, e.g., G. Galfre et al.(1977) Nature 266:55052; Gefter et al Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line.

[0140] Preferred immortal cell lines are mouse myeloma cell lines thatare sensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind to a marker protein, e.g., using a standardELISA assay.

[0141] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-marker protein antibody can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with marker protein tothereby isolate immunoglobulin library members that bind to a markerprotein. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No.WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/20791; Markland et al.PCT International Publication No. WO 92/15679; Breitling et al. PCTInternational Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0142] Additionally, recombinant anti-marker protein antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

[0143] Completely human antibodies are particularly desirable fortherapeutic treatment of human subjects. Such antibodies can be producedusing transgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide corresponding to a marker of the invention. Monoclonalantibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995) Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S.Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016;and U.S. Pat. No. 5,545,806. In addition, companies such as Abgenix,Inc. (Freemont, Calif.), can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

[0144] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1994, Bio/technology12:899-903).

[0145] An anti-marker protein antibody (e.g., monoclonal antibody) canbe used to isolate a marker protein of the invention by standardtechniques, such as affinity chromatography or immunoprecipitation. Ananti-marker protein antibody can facilitate the purification of naturalmarker proteins from cells and of recombinantly produced marker proteinsexpressed in host cells. Moreover, an anti-marker protein antibody canbe used to detect marker protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the marker protein. Anti-marker protein antibodies can beused diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i. e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0146] III. Recombinant Expression Vectors and Host Cells

[0147] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a marker proteinof the invention (or a portion thereof). As used herein, the term“vector” includes a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which includes a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

[0148] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein(e.g., marker proteins, mutant forms of marker proteins, fusionproteins, and the like).

[0149] The recombinant expression vectors of the invention can bedesigned for expression of marker proteins in prokaryotic or eukaryoticcells. For example, marker proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using baculovirus expression vectors)yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

[0150] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

[0151] Purified fusion proteins can be utilized in marker activityassays, (e.g., direct assays or competitive assays described in detailbelow), or to generate antibodies specific for marker proteins, forexample.

[0152] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11 d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET i 1d vector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0153] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to alterthe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., (1 992) NucleicAcids Res. 20:2111-21 1 8). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0154] In another embodiment, the marker protein expression vector is ayeast expression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0155] Alternatively, marker proteins of the invention can be expressedin insect cells using baculovirus expression vectors. Baculovirusvectors available for expression of proteins in cultured insect cells(e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. CellBiol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989)Virology 170:31-39).

[0156] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0157] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0158] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to mRNA corresponding to a KIAA 18 and/or KIAA 96gene. Regulatory sequences operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0159] Another aspect of the invention pertains to host cells into whicha nucleic acid molecule of the invention is introduced, e.g., a KIAA 18and/or KIAA 96 within a recombinant expression vector or a nucleic acidmolecule of the invention containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0160] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a marker protein of the invention can be expressed in bacterialcells such as E. coli, insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

[0161] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0162] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a marker protein or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0163] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a markerprotein. Accordingly, the invention further provides methods forproducing a marker protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding a marker proteinhas been introduced) in a suitable medium such that a marker protein ofthe invention is produced. In another embodiment, the method furthercomprises isolating a marker protein from the medium or the host cell.

[0164] The host cells of the invention can also be used to producenon-human transgenic animals. For example, in one embodiment, a hostcell of the invention is a fertilized oocyte or an embryonic stem cellinto which marker-protein-coding sequences have been introduced. Suchhost cells can then be used to create non-human transgenic animals inwhich exogenous sequences encoding a marker protein of the inventionhave been introduced into their genome or homologous recombinant animalsin which endogenous sequences encoding the marker proteins of theinvention have been altered. Such animals are useful for studying thefunction and/or activity of a marker protein and for identifying and/orevaluating modulators of marker protein activity. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous KIAA 18 and/or KIAA 96 genehas been altered by homologous recombination between the endogenous geneand an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

[0165] A transgenic animal of the invention can be created byintroducing a marker-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene to direct expression of a marker protein to particular cells.Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a transgene of the invention in its genome and/or expressionof mRNA corresponding to a gene of the invention in tissues or cells ofthe animals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding a marker protein can further be bred toother transgenic animals carrying other transgenes.

[0166] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of a gene of the invention into whicha deletion, addition or substitution has been introduced to therebyalter, e.g., functionally disrupt, the gene. The gene can be a humangene, but more preferably, is a non-human homologue of a human KIAA 18and/or KIAA 96. For example, a mouse gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous gene of the invention in the mouse genome. Ina preferred embodiment, the homologous recombination nucleic acidmolecule is designed such that, upon homologous recombination, theendogenous gene of the invention is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector).

[0167] Alternatively, the homologous recombination nucleic acid moleculecan be designed such that, upon homologous recombination, the endogenousgene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous marker protein). In thehomologous recombination nucleic acid molecule, the altered portion ofthe gene of the invention is flanked at its 5′ and 3′ ends by additionalnucleic acid sequence of the gene of the invention to allow forhomologous recombination to occur between the exogenous gene carried bythe homologous recombination nucleic acid molecule and an endogenousgene in a cell, e.g., an embryonic stem cell. The additional flankingnucleic acid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The homologous recombination nucleic acidmolecule is introduced into a cell, e.g., an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced gene hashomologously recombined with the endogenous gene are selected (see e.g.,Li, E. et al. (1992) Cell 69:915). The selected cells can then injectedinto a blastocyst of an animal (e.g., a mouse) to form aggregationchimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987)pp. 113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

[0168] In another embodiment, transgenic non-human animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351 -1355. If a cre/loxPrecombinase system is used to regulate expression of the transgene,animals containing transgenes encoding both the Cre recombinase and aselected protein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

[0169] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0170] IV. Pharmaceutical Compositions

[0171] The nucleic acid molecules of the invention i.e. KIAA 18 and/orKIAA 96, fragments of marker proteins, and anti-marker proteinantibodies (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

[0172] The invention includes methods for preparing pharmaceuticalcompositions for modulating the expression or activity of a polypeptideor nucleic acid corresponding to a marker of the invention. Such methodscomprise formulating a pharmaceutically acceptable carrier with an agentwhich modulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention. Such compositions canfurther include additional active agents. Thus, the invention furtherincludes methods for preparing a pharmaceutical composition byformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention and one or more additionalactive compounds.

[0173] The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which (a) bind to the marker, or (b) have amodulatory (e.g., stimulatory or inhibitory) effect on the activity ofthe marker or, more specifically, (c) have a modulatory effect on theinteractions of the marker with one or more of its natural substrates(e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d)have a modulatory effect on the expression of the marker. Such assaystypically comprise a reaction between the marker and one or more assaycomponents. The other components may be either the test compound itself,or a combination of test compound and a natural binding partner of themarker.

[0174] The test compounds of the present invention may be obtained fromany available source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994,J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam, 1997, AnticancerDrug Des. 12:145).

[0175] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g. inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0176] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0177] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a fragment of a marker protein or an anti-markerprotein antibody) in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0178] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0179] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0180] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0181] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0182] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0183] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0184] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0185] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i. e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0186] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0187] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0188] V. Computer Readable Means and Arrays

[0189] Computer readable media comprising a marker(s) of the presentinvention is also provided. As used herein, “computer readable media”includes a medium that can be read and accessed directly by a computer.Such media include, but are not limited to: magnetic storage media, suchas floppy discs, hard disc storage medium, and magnetic tape; opticalstorage media such as CD-ROM; electrical storage media such as RAM andROM; and hybrids of these categories such as magnetic/optical storagemedia. The skilled artisan will readily appreciate how any of thepresently known computer readable mediums can be used to create amanufacture comprising computer readable medium having recorded thereona marker of the present invention.

[0190] As used herein, “recorded” includes a process for storinginformation on computer readable medium. Those skilled in the art canreadily adopt any of the presently known methods for recordinginformation on computer readable medium to generate manufacturescomprising the markers of the present invention.

[0191] A variety of data processor programs and formats can be used tostore the marker information of the present invention on computerreadable medium. For example, the nucleic acid sequence corresponding tothe markers can be represented in a word processing text file, formattedin commercially-available software such as WordPerfect and MicroSoftWord, or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. Any number ofdataprocessor structuring formats (e.g., text file or database) may beadapted in order to obtain computer readable medium having recordedthereon the markers of the present invention.

[0192] By providing the markers of the invention in computer readableform, one can routinely access the marker sequence information for avariety of purposes. For example, one skilled in the art can use thenucleotide or amino acid sequences of the invention in computer readableform to compare a target sequence or target structural motif with thesequence information stored within the data storage means. Search meansare used to identify fragments or regions of the sequences of theinvention which match a particular target sequence or target motif.

[0193] The invention also includes an array comprising a marker(s) ofthe present invention. The array can be used to assay expression of oneor more genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 8600 genes can besimultaneously assayed for expression. This allows a profile to bedeveloped showing a battery of genes specifically expressed in one ormore tissues.

[0194] In addition to such qualitative determination, the inventionallows the quantitation of gene expression. Thus, not only tissuespecificity, but also the level of expression of a battery of genes inthe tissue is ascertainable. Thus, genes can be grouped on the basis oftheir tissue expression per se and level of expression in that tissue.This is useful, for example, in ascertaining the relationship of geneexpression between or among tissues. Thus, one tissue can be perturbedand the effect on gene expression in a second tissue can be determined.In this context, the effect of one cell type on another cell type inresponse to a biological stimulus can be determined. Such adetermination is useful, for example, to know the effect of cell-cellinteraction at the level of gene expression. If an agent is administeredtherapeutically to treat one cell type but has an undesirable effect onanother cell type, the invention provides an assay to determine themolecular basis of the undesirable effect and thus provides theopportunity to co-administer a counteracting agent or otherwise treatthe undesired effect. Similarly, even within a single cell type,undesirable biological effects can be determined at the molecular level.Thus, the effects of an agent on expression of other than the targetgene can be ascertained and counteracted.

[0195] In another embodiment, the array can be used to monitor the timecourse of expression of one or more genes in the array. This can occurin various biological contexts, as disclosed herein, for exampledevelopment and differentiation, disease progression, in vitroprocesses, such a cellular transformation and senescence, autonomicneural and neurological processes, such as, for example, pain andappetite, and cognitive functions, such as learning or memory.

[0196] The array is also useful for ascertaining the effect of theexpression of a gene on the expression of other genes in the same cellor in different cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

[0197] The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and diseased cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

[0198] VI. Predictive Medicine

[0199] The present invention pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, pharmacogeneticsand monitoring clinical trials are used for prognostic (predictive)purposes to thereby treat an individual prophylactically. Accordingly,one aspect of the present invention relates to diagnostic assays fordetermining marker protein and/or nucleic acid expression as well asmarker protein activity, in the context of a biological sample (e.g.,blood, serum, cells, tissue) to thereby determine whether an individualis afflicted with a disease or disorder, or is at risk of developing adisorder, associated with increased or decreased marker proteinexpression or activity. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with marker protein, nucleic acidexpression or activity. For example, the number of copies of a markergene can be assayed in a biological sample. Such assays can be used forprognostic or predictive purposes to thereby phophylactically treat anindividual prior to the onset of a disorder (e.g., prostate cancer)characterized by or associated with marker protein, nucleic acidexpression or activity.

[0200] Another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs, compounds) on the expression oractivity of marker in clinical trials.

[0201] These and other agents are described in further detail in thefollowing sections.

[0202] 1. Diagnostic Assays

[0203] An exemplary method for detecting the presence or absence ofmarker protein or nucleic acid of the invention in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting the protein or nucleic acid (e.g., mRNA, genomic DNA) thatencodes the marker protein such that the presence of the marker proteinor nucleic acid is detected in the biological sample. A preferred agentfor detecting mRNA or genomic DNA corresponding to a marker gene orprotein of the invention is a labeled nucleic acid probe capable ofhybridizing to a mRNA or genomic DNA of the invention. Suitable probesfor use in the diagnostic assays of the invention are described herein.

[0204] A preferred agent for detecting marker protein is an antibodycapable of binding to marker protein, preferably an antibody with adetectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i. e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect marker mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of marker mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of marker protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of marker genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of marker protein include introducing into a subject a labeledanti-marker antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

[0205] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject. A preferred biological sample is aserum sample isolated by conventional means from a subject.

[0206] In another embodiment, the methods further involve obtaining acontrol biological sample (e.g., non-prostate cancer cells sample) froma control subject, contacting the control sample with a compound oragent capable of detecting marker protein, mRNA, or genomic DNA, suchthat the presence of marker protein, mRNA or genomic DNA is detected inthe biological sample, and comparing the presence of marker protein,mRNA or genomic DNA in the control sample with the presence of markerprotein, mRNA or genomic DNA in the test sample.

[0207] The invention also encompasses kits for detecting the presence ofmarker in a biological sample. For example, the kit can comprise alabeled compound or agent capable of detecting marker protein or mRNA ina biological sample; means for determining the amount of marker in thesample; and means for comparing the amount of marker in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect marker protein or nucleic acid.

[0208] 2. Prognostic Assays

[0209] The diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing a diseaseor disorder associated with aberrant marker expression or activity. Asused herein, the term “aberrant” includes a marker expression oractivity which deviates from the wild type marker expression oractivity. Aberrant expression or activity includes increased ordecreased expression or activity, as well as expression or activitywhich does not follow the wild type developmental pattern of expressionor the subcellular pattern of expression. For example, aberrant markerexpression or activity is intended to include the cases in which amutation in the marker gene causes the marker gene to be under-expressedor over-expressed and situations in which such mutations result in anon-functional marker protein or a protein which does not function in awild-type fashion, e.g., a protein which does not interact with a markerligand or one which interacts with a non-marker protein ligand.

[0210] The assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with amisregulation in marker protein activity or nucleic acid expression,such as prostate cancer. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adisorder associated with a misregulation in marker protein activity ornucleic acid expression, such as prostate cancer. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant marker expression or activity in which a testsample is obtained from a subject and marker protein or nucleic acid(e.g., mRNA or genomic DNA) is detected, wherein the presence of markerprotein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant markerexpression or activity. As used herein, a “test sample” includes abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., blood), cell sample, ortissue (e.g., skin).

[0211] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with increased or decreased marker expression or activity.For example, such methods can be used to determine whether a subject canbe effectively treated with an agent for a disorder such as prostatecancer. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with increased or decreased marker expression oractivity in which a test sample is obtained and marker protein ornucleic acid expression or activity is detected (e.g., wherein theabundance of marker protein or nucleic acid expression or activity isdiagnostic for a subject that can be administered the agent to treat adisorder associated with increased or decreased marker expression oractivity).

[0212] The methods of the invention can also be used to detect geneticalterations in a marker gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inmarker protein activity or nucleic acid expression, such as prostatecancer. In preferred embodiments, the methods include detecting, in asample of cells from the subject, the presence or absence of a geneticalteration characterized by at least one of an alteration affecting theintegrity of a gene encoding a marker-protein, or the mis-expression ofthe marker gene. For example, such genetic alterations can be detectedby ascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a marker gene; 2) an addition of one or morenucleotides to a marker gene; 3) a substitution of one or morenucleotides of a marker gene, 4) a chromosomal rearrangement of a markergene; 5) an alteration in the level of a messenger RNA transcript of amarker gene, 6) aberrant modification of a marker gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a marker gene, 8)a non-wild type level of a marker-protein, 9) allelic loss of a markergene, and 10) inappropriate post-translational modification of amarker-protein. As described herein, there are a large number of assaysknown in the art which can be used for detecting alterations in a markergene. A preferred biological sample is a tissue or blood sample isolatedby conventional means from a subject.

[0213] In certain embodiments, detection of the alteration involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.,U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which canbe particularly useful for detecting point mutations in the marker-gene(see Abravaya et al. (1995) Nucleic Acids Res.23:675-682). This methodcan include the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a marker gene under conditions such thathybridization and amplification of the marker-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

[0214] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0215] In an alternative embodiment, mutations in a marker gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0216] In other embodiments, genetic mutations in a marker gene or agene encoding a marker protein of the invention can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in marker can be identified in two dimensional arrayscontaining light-generated DNA probes as described in Cronin, M. T. etal. supra. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0217] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the markergene and detect mutations by comparing the sequence of the sample markerwith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

[0218] Other methods for detecting mutations in the marker gene or geneencoding a marker protein of the invention include methods in whichprotection from cleavage agents is used to detect mismatched bases inRNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type marker sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with SI nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

[0219] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in marker cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on amarker sequence, e.g., a wild-type marker sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

[0220] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in marker genes or genes encoding amarker protein of the invention. For example, single strand conformationpolymorphism (SSCP) may be used to detect differences in electrophoreticmobility between mutant and wild type nucleic acids (Orita et al. (1989)Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res.285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).Single-stranded DNA fragments of sample and control marker nucleic acidswill be denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0221] In yet another embodiment the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0222] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl.Acad. Sci USA 86:6230). Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0223] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

[0224] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose subjectsexhibiting symptoms or family history of a disease or illness involvinga marker gene.

[0225] Furthermore, any cell type or tissue in which marker is expressedmay be utilized in the prognostic assays described herein.

[0226] 3. Monitoring of Effects During Clinical Trials

[0227] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a marker protein (e.g., the modulation ofprostate cancer) can be applied not only in basic drug screening, butalso in clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase markergene expression, protein levels, or upregulate marker activity, can bemonitored in clinical trials of subjects exhibiting decreased markergene expression, protein levels, or downregulated marker activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease marker gene expression, protein levels, ordownregulate marker activity, can be monitored in clinical trials ofsubjects exhibiting increased marker gene expression, protein levels, orupregulated marker activity. In such clinical trials, the expression oractivity of a marker gene, and preferably, other genes that have beenimplicated in, for example, a marker-associated disorder (e.g., prostatecancer) can be used as a “read out” or markers of the phenotype of aparticular cell.

[0228] For example, and not by way of limitation, genes, includingmarker genes and genes encoding a marker protein of the invention, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates marker activity (e.g., identified ina screening assay as described herein) can be identified. Thus, to studythe effect of agents on marker-associated disorders (e.g. prostatecancer), for example, in a clinical trial, cells can be isolated and RNAprepared and analyzed for the levels of expression of marker and othergenes implicated in the marker-associated disorder, respectively. Thelevels of gene expression (e.g., a gene expression pattern) can bequantified by northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofmarker or other genes. In this way, the gene expression pattern canserve as a marker, indicative of the physiological response of the cellsto the agent. Accordingly, this response state may be determined before,and at various points during treatment of the individual with the agent.

[0229] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) including the stepsof (i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression of amarker protein, mRNA, or genomic DNA in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of themarker protein, mRNA, or genomic DNA in the post-administration samples;(v) comparing the level of expression or activity of the marker protein,mRNA, or genomic DNA in the pre-administration sample with the markerprotein, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of marker tohigher levels than detected, i.e., to increase the effectiveness of theagent. Alternatively, decreased administration of the agent may bedesirable to decrease expression or activity of marker to lower levelsthan detected, i.e. to decrease the effectiveness of the agent.According to such an embodiment, marker expression or activity may beused as an indicator of the effectiveness of an agent, even in theabsence of an observable phenotypic response.

[0230] 4. Methods of Treatment

[0231] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk for (or susceptibleto) a disorder or having a disorder associated with aberrant markerexpression or activity. With regards to both prophylactic andtherapeutic methods of treatment, such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics. “Pharmacogenomics”, as used herein, includes theapplication of genomics technologies such as gene sequencing,statistical genetics, and gene expression analysis to drugs in clinicaldevelopment and on the market. More specifically, the term refers thestudy of how a subject's genes determine his or her response to a drug(e.g., a subject's “drug response phenotype”, or “drug responsegenotype”.) Thus, another aspect of the invention provides methods fortailoring an individual's prophylactic or therapeutic treatment witheither the marker molecules of the present invention or markermodulators according to that individual's drug response genotype.Pharmacogenomics allows a clinician or physician to target prophylacticor therapeutic treatments to subjects who will most benefit from thetreatment and to avoid treatment of subjects who will experience toxicdrug-related side effects.

[0232] 5. Prophylactic Methods

[0233] In one aspect, the invention provides a method for preventing ina subject, a disease or condition (e.g., prostate cancer) associatedwith increased or decreased marker expression or activity, byadministering to the subject a marker protein or an agent whichmodulates marker protein expression or at least one marker proteinactivity. Subjects at risk for a disease which is caused or contributedto by increased or decreased marker expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe differential marker protein expression, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.Depending on the type of marker aberrancy (e.g., increase or decrease inexpression level), for example, a marker protein, marker protein agonistor marker protein antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

[0234] 6. Therapeutic Methods

[0235] Another aspect of the invention pertains to methods of modulatingmarker protein expression or activity for therapeutic purposes.Accordingly, in an exemplary embodiment, the modulatory method of theinvention involves contacting a cell with a marker protein or agent thatmodulates one or more of the activities of a marker protein activityassociated with the cell. An agent that modulates marker proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring target molecule of a marker protein(e.g., a marker protein substrate), a marker protein antibody, a markerprotein agonist or antagonist, a peptidomimetic of a marker proteinagonist or antagonist, or other small molecule. In one embodiment, theagent stimulates one or more marker protein activities. Examples of suchstimulatory agents include active marker protein and a nucleic acidmolecule encoding marker protein that has been introduced into the cell.In another embodiment, the agent inhibits one or more marker proteinactivities. Examples of such inhibitory agents include antisense markerprotein nucleic acid molecules, anti-marker protein antibodies, andmarker protein inhibitors. These modulatory methods can be performed invitro (e.g., by culturing the cell with the agent) or, alternatively, invivo (e.g., by administering the agent to a subject). As such, thepresent invention provides methods of treating an individual afflictedwith a disease or disorder characterized by aberrant expression oractivity of a marker protein or nucleic acid molecule. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., upregulates or downregulates) markerprotein expression or activity. In another embodiment, the methodinvolves administering a marker protein or nucleic acid molecule astherapy to compensate for reduced or aberrant marker protein expressionor activity.

[0236] Stimulation of marker protein activity is desirable in situationsin which marker protein is abnormally downregulated and/or in whichincreased marker protein activity is likely to have a beneficial effect.For example, stimulation of marker protein activity is desirable insituations in which a marker is downregulated and/or in which increasedmarker protein activity is likely to have a beneficial effect. Likewise,inhibition of marker protein activity is desirable in situations inwhich marker protein is abnormally upregulated and/or in which decreasedmarker protein activity is likely to have a beneficial effect.

[0237] 7. Pharmacogenomics

[0238] The marker protein and nucleic acid molecules of the presentinvention, as well as agents, or modulators which have a stimulatory orinhibitory effect on marker protein activity (e.g., marker geneexpression) as identified by a screening assay described herein can beadministered to individuals to treat (prophylactically ortherapeutically) marker-associated disorders (e.g., prostate cancer)associated with aberrant marker protein activity. In conjunction withsuch treatment, pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) may be considered. Differences in metabolismof therapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a marker molecule or markermodulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a marker molecule or marker modulator.

[0239] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum, M. etal. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 and Linder,M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0240] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of subjects taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

[0241] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drugs target is known (e.g., amarker protein of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

[0242] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0243] Alternatively, a method termed the “gene expression profiling”,can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a drug (e.g., amarker molecule or marker modulator of the present invention) can givean indication whether gene pathways related to toxicity have been turnedon.

[0244] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with amarker molecule or marker modulator, such as a modulator identified byone of the exemplary screening assays described herein.

[0245] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of a Marker cDNA

[0246] (i) Methods and Materials

[0247] (a) Cell Cultures

[0248] Human prostatic cancer cell lines LNCaP, DU-145, PC-3, andTsu-prl were obtained from ATCC. LNCaP cancer cells were maintained inhumidified atmosphere of 5% CO₂ in air in RPMI 1640 medium supplementedwith 10% fetal calf serum (Life Technologies, Inc, Rockville, Md.), 3 mML-glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin. Otherlines were maintained in DMEM containing 3 mM L-glutamine, 100 μg/mlstreptomycin, 100 units/ml penicillin, and 10% FCS in humidifiedatmosphere of 5% CO₂. To examine the effects of steroids, cells werecultured in RPMI 1640 medium containing 5% FCS treated with dextrancoated charcoal (Hyclone, Logan, Utah) for 24 hs before treatment. Cellswere grown in the absence or presence of 10 nM DHT for 0, 2, 4, 6, 12,24, 48, and 72 hs. They were collected and frozen at each time point.Two hundred μl of medium were collected from each flask for PSA assay.

[0249] (b) Cell Growth Assays

[0250] To verify the effect of DHT on the growth of LNCaP cells, cellsat 3,000 cells/well were plated in 96-well plates for 24 hs beforetreatment with DHT. After 72 hs, MTT was added to each well andincubated at 37° C. for four hs. At the end of incubation, thesupernatant was removed and 100 μl DMSO was added to each well todissolve the cells. Plates were subsequently read in a plate reader at570 nM.

[0251] (c) PSA ELISA

[0252] Quantification of PSA was performed using an ELISA. Briefly, a96-well Nunc plate was coated with 100 μl of goat anti-PSA (1 μg/ml,Scripps laboratory, San Diego, Calif.) overnight at 4° C. The plate waswashed with water three times and incubated with 100 μl of blockingbuffer (PBS, 0.05% Tween 20, 1 μM EDTA, 0.25% BSA, and 0.05% NaN₃) for 1h at room temperature. The plate was washed three times with water andincubated with 1:1 mixture of mouse anti-human PSA and Eu-labeledanti-mouse IgG (10 ng/antibody each/well for 1½ hs at RT). The plate wasthen washed four times with water. 100 μl of Delfia Enhancement Solution(PerkinElmer Wallac Inc (Norton, Ohio) was added to the plate was readusing a Victor reader according to the manufacturer's instruction.

[0253] (d) RNA extraction and preparation

[0254] Total RNA was isolated from LNCaP cells using the Qiagen RneasyMidi Kit following the manufacturer's recommendations. For polyA (+)selection, the Promega PolyATract kit was used according tomanufacturer's procedures. Briefly, LNCaP cells were collected bycentrifugation and the RNA isolated using the buffers and recommendedprocedures from the Qiagen kit. Following RNA extraction, all sampleswere frozen at −80° C. One microgram of poly A(+) RNA was used astemplate for synthesis of double-stranded cDNA using the GibcoBRL cDNAsynthesis kit, with an oligo dT primer incorporating a T7 RNA polymerasepromoter (10 minutes at 70° C. for priming, 65 minutes at 37° C. forfirst strand synthesis with Superscript II RT, followed by 150 min at15.8° C. for second strand synthesis with E. coli ligase, E. colipolymerase, and RNAse H). The double-stranded cDNA was purified by SolidPhase Reversible Immobilization (SPRI) using the methods described by DeAngelis et al. using Perseptives paramagnetic beads (See, De Angelis etal. (1995) Nuc. Acid Res. 23: 4742-4743.) Approximately 50 ng ofdouble-stranded cDNA was used as template for in vitro transcription tomake labeled cRNA (16 hours at 37° C., Epicenter T7 RNA polymerase, EnzoLaboratories bio-11-CTP, bio-11-UTP). The cRNA was purified by SPRIusing paramagnetic beads (Bangs Laboratories), and total molarconcentration was determined from the absorbance at 260. Prior tohybridization, 10 ug of labeled cRNA was fragmented randomly to anaverage length of approximately 50 bases by heating at 94° C. in 40 mMTris-acetate pH 8.1, 100 mM potassium acetate, and 30 mM magnesiumacetate, for 35 minutes.

[0255] For material made directly from cellular RNA, cytoplasmic RNA wasextracted from cells by the method of Favaloro et al. ((1980) MethodsEnzymol. 65: 718-749), and poly (A) RNA was isolated with an oligo dTselection step (Promega PolyA tract mRNA Isolation System IV, Madison,Wis.).

[0256] (e) Chip Hybridization and Analysis

[0257] Affymetrix Genechip™ technology was used to monitor theexpression of about 6000 full-length human genes in response to anatural androgen DHT in LNCaP cells. FIG. 2 illustrates the generalscheme used for sample preparation, hybridization, and analysis.Hybridization cocktail was made using 10 μg of fragmented cRNA, 2×MESbuffer with BSA, herring sperm DNA, control prokaryotic transcripts forinternal control, and biotinylated control oligo 948 (for chip qualitycontrol). DEPC-water was added to bring the volume to 200 μl. Prior tohybridization, the hybridization cocktails were heated to 99° C. for 10minutes, and then 37° C. for an additional 10 minutes before loadinginto Hu6800FL arrays (Affymetrix GeneChips™). The Hu680OFl array iscomprised of 6800 known full-length genes, about 250,000 25-meroligonucleotide probes with 20 probe pairs per gene. Array hybridizationproceeded overnight at 45° C. with 50 rpm. Following hybridization, thearrays were washed and stained using the manufacturer's recommendationsand procedures. (Affymetrix Expression Analysis Technical Manual).Non-stringent wash buffer (20×SSPE, 1.0 ml of 10% Tween 20, and water)at 25° C., and stringent wash buffer (20×SSPE, 5M NaCl, 10% Tween 20,and water) at 50° C. were used for the wash steps. The arrays were thenstained with strepavidin-conjugated phycoerythrin (SAPE, MolecularProbes), followed by biotinylated anti-strepavidin and a second round ofSAPE for signal amplification at 25 ° C. Each stain step was done for 10minutes. All arrays were then scanned using the HP Genearray Scanner andthe resulting fluorescence emmisions were collected and quantified usingAffymetrix Genechip software. Within the software, the signalintensities for all the probes on each array were calculated from thescanned image, and the appropriate probe array algorithm was applied todetermine the expression levels (average difference) for each gene.Average differences for all genes were converted into mRNA frequencyestimates (in molecules per million) based on the standard spike-incontrol transcripts.

[0258] (f) Data Filtering and Statistics

[0259] Initial data was reduced by filtering for all genes called“present” by GeneChip™. A two-way ANOVA was then performed on thereplicate data for each of these genes in the statistical computingpackage S-plus. The potential effects of two experimental factors(treatment and time) and the interaction of both factors on theexpression level were evaluated in the analysis of variance model, andthe p-values for the main effects (P_(treatment), P_(time)) and for theinteraction(P_(interact)) were obtained. Only those genes that werestatistically significant (p-value <=0.05) for the treatment factorand/or the interaction were considered for the time being. First, theaverage was taken for baseline and experimental replicate mRNAfrequencies of the 705 genes that passed this p-value criterion. Averagefrequencies obtained for each gene were then standardized across allsamples to have a mean of zero and a standard deviation of one. Amodified version of the original self-organizing map (SOM) algorithmdeveloped by Kohonen et al (Self-Organizing Maps, Second ExtendedEdition edition, Vol. 30. New York, 1997), created using the MATLABtoolbox, was then applied to the standardized expression values togenerate a 6 by 6 matrix of 36 clusters (Tamayo et al. (1999) Proc.Natl. Acad. Sci. USA. 96: 2907-2912). Several public databases such asGenecards and Swiss-Prot were used for gene annotation (See e.g., Rebhanet al. GeneCards: encyclopedia for genes, proteins and diseases.Weizmann Institute of Science, Bioinformatics Unit and Genome Center(Rehovot, Israel), 1997. World Wide Web URL:http://bioinfo.weizmann.ac.il/cards, and Appel et al. (1994).A newgeneration of information retrieval tools for biologists: the example ofthe ExPASy WWW server.Trends Biochem. Sci. 19:258-260 World Wide WebURL: http://www.expasy.ch/sprot/).

[0260] (g) Quantitative Taqman RT-PCR

[0261] The same total RNA samples used for the GeneChip experiments wereanalyzed using a Taqman® EZ RT-PCR kit. (PE Applied Biosystems) toconfirm gene expression changes. Total RNA samples were diluted to aconcentration of 50 ng/ul and a total of 50 ng was used for eachreaction. Primers and florescence probes for PSA and KIAA18 and KIAA96were designed using the Primer Express software and were chosen basedupon the manufacturer's recommendations for primer selection. Theprimers used were of 100 uM concentration and were as follows: (a) PSA-F(forward primer) CGTGGCCAACCCCTGA (SEQ ID NO: 1), PSA-R (reverse primer)CTTGGCCTGGTCATTTCCAA (SEQ ID NO: 2), and PSA-P (probe)CACCCCTATCAACCCCCTATTGTAGTAAACTTGGA (SEQ ID NO: 3). (b) KIAA 18-F(forward primer) CAAGATCCTTCCTTCAACCCC (SEQ ID NO: 4), KIAA 18-R(reverse primer) TGGCACCTGGAATGACAAGA (SEQ ID NO: 5), KIAA 18-P (probe)AGCTCCCATCTCATTTCCAGAAAGGCTCAT (SEQ ID NO: 6); and (d) KIAA 96-F(forward primer) GTCATGTGTCTGAGGTGACGGA (EQ ID NO: 7), KIAA 96-R(reverse primer) TGAAGAAACAGTGACCACAGCAAT (SEQ ID NO: 8), and KIAA 96-P(probe) TGGTCCTGTAATTCAGAGAGTGGGCACATCACC (SEQ ID NO: 9).

[0262] Samples were prepared using a reagent mix of manufacturersupplied RT-PCR components [(5×TaqMan EZ Buffer, manganese acetate (25mM), dATP (10mM), dCTP (10 mM), dGTP (10 mM) and dUTP (20 mM), rTth DNApolymerase (2.5 U/μl), AmpErase UNG (1 U/μl), primers (finalconcentration 1 μM) and RNA (50 ng)], following manufacturer'srecommendations. In addition, GAPDH control samples for standard curvegeneration and subsequent quantitation of sample RNA was prepared.Primers and probe for GAPDH were included in the kit (GAPDH forward andreverse primers 10 μM, GAPDH probe 5 μM). β-actin was also used forstandard curve generation, and dilutions were made for both genes thatranged from 5×10 ⁶ copies to 5×10¹ copies. The assay was performed on aPerkin-Elmer/Applied Biosystems 7700 Prism, and the PCR cyclingparameters were chosen based on the manufacturer's recommendations. RNAof samples were normalized to GAPDH and β-actin and was quantified.

[0263] (h) Western Blot Analysis

[0264] To demonstrate that the protein production of KIAA (e.g., KIAA 18and/or KIAA 96) can be regulated by androgen, Western blot analysis canbe performed. For Western blot analysis, LNCaP cells can be plated in6-well plate at 1×10⁶ cells/well in charcoal stripped serum containingmedium. Cells can be treated with a suitable amount of androgen, e.g.,10 nM DHT and harvested at designated time. Cells can be harvested inMPER reagent (Pierce, Rockford, Ill.) containing 400 mM NaCl. Proteincan be quantified by Bradford method (Bradford (1976) Anal. Bioch. 72:248-254). A suitable amount of protein, e.g., 30 μg of protein can beelectrophoresed on a 12% SDS-PAGE gel and transferred to a PVDF membraneusing a Bio Rad liquid transfer apparatus. The PVDF membrane can beincubated in TBST (TBS with 0.1% Tween-20) with 3% milk for 15 minutesbefore the addition of the first antibody, e.g., rabbit anti-KIAAantibody (anti-KIAA 18, or anti-KIAA 96 antibody). After overnightincubation, the PVDF membrane can be washed 3 times with TBST andincubated with a second antibody, anti-rabbit-IgG coupled withhorseradish peroxidase (Transduction Labs) for one hour. The PVDFmembrane can then be washed 3 times with TBST and protein can bedetected by using an enhanced chemiluminescence detection system(Pierce).

[0265] (i) Tissue Microarray Construction and Analysis

[0266] To investigate the role of KIAA, e.g., KIAA 18 or KIAA 96 insolid tumors, tissue microarray analysis can be performed on multiplehuman normal (i.e., control samples) and prostate diseased specimens(Clinomics, Inc.). Following fixation in 10% neutral buffered formalin,tissues can be selected, trimmed, and placed in a processing cassette.The cassette can then placed in a processing basket on a ShandonHypercenter™ tissue processor in which the tissues can be exposed to aseries of buffers over a 16 hour processing cycle (10% Neutral Bufferedformalin, 70%, 95%, 100% ethanol, xylene, and melted paraffin embeddingmedia). All steps should be carried out under vacuum at 40° C. exceptfor the paraffin steps which should be at 58° C. Following processing,the tissues can be removed from the cassettes and embedded in paraffinblocks. The resulting blocks can be sectioned at 5 μm and mounted onglass slides. The slides can be heated at 58° C. for 30 minutes prior tostaining. Antibody α-KIAA (e.g., anti-KIAA 18 or anti-KIAA 96) can betitered to a suitable dilution, e.g., 1:150 dilution using DAKO®Antibody Diluent. Staining of test specimen can be performed employingHIER in pH 6.0 citrate buffer with no pretreatment. Tissues can then bestained using the Ventana ES® Automated Immunohistochemistry Stainer,involving the use of a standard indirect immunoperoxidase protocol with3,3 ′-diaminobenzidine as a chromagen. Grading of theimmunohistochemical staining is based on the intensity of thecytoplasmic staining of the epithelial components of both the tumor andthe normal tissues. The strength of the staining can be scored using a1+ to 4+ scale, 1+ indicating faint staining and 4+ indicating strongeststaining (appearing as dark brown staining). A score of 0 indicated nostaining.

[0267] (j) Transient Transfection of COS cells

[0268] To determine the effect of KIAA, (e.g., KIAA 18 and/or KIAA 96)on the transcriptional activity of androgen receptor (AR), COS-1 cellscan be transiently transfected with a reporter construct containingandrogen receptor response element along with an expression vectorencoding KIAA 18 or KIAA 96. COS-1 cells can be plated in 6-well platesat a density of 2×10⁵ cells per well in 2-ml phenol red-free DMEMcontaining 10% charcoal-stripped fetal bovine serum. The next morning,medium can be replaced with 2-ml DMEM. Indicated amount of DNA in 100 μlof DMEM can be mixed with 6 μl of PLUS reagent (Gibco) and incubated atroom temperature while 4 μl of lipofectamine can be mixed with 100 μl ofDMEM. After 30 min of incubation, the two mixtures can be combinedtogether and added dropwise to each well. After incubation with DNA for4 hours, 2 ml of phenol red-free DMEM containing 10% charcoal-strippedfetal bovine serum can be added and cells treated with indicatedchemicals for additional 24 hours before being harvested.

[0269] (k) Luciferase Assay

[0270] Luciferase activity can be determined using Promega's Steady-GloLuciferase Assay System. Briefly, after 24 hours of treatment, cells canbe harvested by scraping in 1 ml of PBS. A suitable amount of protein,e.g., 5 μg from each sample in a total of 100 μl PBS can be mixed with100 μl of Stable-Glo reagent (Promega), and luminescence can bedetermined in a luminometer (Wallac, 1450 MicroBeth Counter) after 5min.

[0271] (ii) Results

[0272] DHT stimulates the Growth of LNCaP Cells and PSA Production

[0273] LNCaP cells are widely used as tumor models because they maintainresponsiveness to androgen (Horoszewicz et al. (1983). Cancer Res 43:1809-1818). For example, their ability to proliferate, to expressdifferentiated secretory function, and to control processes such aslipid synthesis and accumulation, all remain androgen responsive. Toascertain whether LNCaP in the present culture conditions could be usedto examine androgen-regulated genes, the response of LNCaP to androgentreatment was tested using the procedures described in sections (a-c).Cell growth and PSA production were studied.

[0274]FIG. 1A shows that the growth of LNCaP cells was stimulated by anatural androgen DHT in a dose-dependent manner. 10 nM DHT was chosenfor the rest of the experiments because of its robust growth-stimulatoryeffect. PSA is a widely used prostate marker and was therefore tested inthe present study prior to the microarray experiment. In response to DHTtreatment, PSA production was increased in a dose-dependent manner (FIG.1B). PSA signal was detected as early as 12 hs and the maximal level wasobserved at about 48 hs. These results demonstrated that LNCaP areresponsive to DHT

[0275] Genechip Hybridization and Analysis

[0276] Affymetrix Genechip™ technology was used to monitor theexpression of about 6000 full-length human genes in response to anatural androgen DHT in LNCaP cells. FIG. 2 illustrates the generalscheme used for sample preparation, hybridization, and analysis and thedetails of hybridization are described in section (e). To obtainreliable data, total RNA was prepared in duplicate from LNCaP cellstreated or not with DHT for 0, 2, 4, 6, 12, 24, 48, and 72 hs asdescribed in section (d). CRNAs were prepared and hybridized also induplicate to Affymetrix chips. Therefore a set of biological replicatesfor a total of 30 samples were generated for each experiment to ensurereproducibility. Only those genes that were called “present” in eitherthe baseline or the experiment in at least one time point and in eitherreplicate passed the initial data reduction filter. Out of about 6000genes represented on the chip, 4491 passed this initial filter (75%).

[0277] Statistical Analysis of Replicates

[0278] To assess reproducibility, the coefficient of variation (CV) tothe mean frequencies of two replicates at each time point were compared.The results showed that across all genes, CV varied between 25 and 35%(data not shown). Based on the experimental design, a two-way analysisof variance (ANOVA) was used to determine the statistical significanceof the ˜4500 gene expression changes. The results based on a 95%significance level show that 200 genes were significant due to androgentreatment alone, 431 genes were significant due to an interaction ofandrogen treatment and time, and 74 genes were significant due to boththe treatment factor and the interaction. Only androgen-regulated geneswere identified, the 242 genes that were significantly modulated due totime alone were not considered.

[0279] Rapid Classification of Expression Profiles using Self-OrganizingMaps

[0280] For rapid classification and to understand the potential functionof candidate genes, expression profiles of the 705 genes found to beregulated by androgen and/or an interaction between androgen and time byANOVA analysis were clustered using an adaptation of the self-organizingmap (SOM) algorithm developed by c and Tamayo et al. (supra), mRNAfrequencies of each gene were averaged within treatment/time subgroups,and the averaged frequencies over all subgroups were standardized suchthat the mean of the averaged frequency was set to zero, and thestandard deviation equal to one. Based on standardized mRNA frequenciesfor each gene, a 6 by 6 matrix of 36 clusters was generated andvisualized.

[0281] Identification of Androgen-Regulated Genes

[0282] For rapid classification and to understand the potential functionof candidate genes, expression profiles of the 705 genes found to beregulated by androgen and/or an interaction between androgen and time byANOVA analysis were clustered using an adaptation of the self-organizingmap (SOM) algorithm developed by Kohonen and Tamayo et al. (Supra). Theresults showed that Cluster (1,1) included genes that shared a similarpattern of induced expression upon androgen treatment, while cluster (6,6) included genes that had a pattern of repressed expression uponandrogen treatment. Genes that are induced in response to androgenclustered together in Cluster (1,1) and included prostate specificantigen (PSA), the most widely used diagnostic marker for prostatecancer. Elevated PSA levels are often detected when cancer is present.In response to androgen treatment, PSA expression (p_(treatment)=0.0000,p_(time)=0.8682, p_(interact)=0.3282) increased 3-fold relative tocontrol at 12 hours, and maintained its high expression through 72 hourswhere it was induced approximately 4-fold (FIG. 3A).

[0283] In response to androgen, the transglutaminase, KIAA 18 alsoshared the same a Cluster (1,1) pattern of induced expression as PSA.KIAA 18 expression increased after androgen treatment(p_(treatment)=0.0014, p_(time)=0.1346, p_(interact)=0.2121). Itsinduction began at 12 hours, and at 24 hours it is over-expressed 2-foldrelative to control (FIG. 3B designated “K18”). Like PSA, KIAA 18maintained its over-expression in response to androgen through 72 hours,at which point it is still induced 3-fold relative to baseline.

[0284] In contrast to PSA, the serine/threonine kinase KIAA 96 was downregulated in response to androgen in LNCaP. Genes that fell withinCluster (6,6) shared a pattern of repressed expression relative tobaseline. One of the genes was Prostatic Acid Phosphatase (PAcP), whichlike PSA, is another prostate-specific antigen. PacP has previously beenshown to be suppressed by DHT in LNCaP (Lin et al. (2000) Cell Biol.Int. 24: 681-689). KIAA 96 produced a Cluster (6,6) pattern(p_(treatment)=0.0000, p_(time)=0.0015, p_(interact)=0.0080). Itsexpression, while consistently repressed relative to control throughoutthe time-course, was most significantly altered at 24 hours where it wasdown 6-fold relative to baseline (FIG. 3C designated “K96”).

[0285] Quantitative RT-PCR Analysis of RNA Samples

[0286] Quantitative RT-PCR was also used to confirm the gene expressionchanges from the GeneChip analysis as described in section (g). Theresults for qualitative RT-PCR are shown in FIG. 4A, B and Cdemonstrating the changes in RNA levels for PSA and KIAA 18and KIAA96.

[0287] In summary, these results show that KIAA 18 and KIAA 96 werefound to be androgen-regulated. KIAA 18 is a novel member of thetransglutaminase-like superfamily. Transglutaminase (TG) catalyzes theacyl transfer reaction between peptide-bound glutamine residues andprimary amine groups. TG may be associated with cell growth regulationduring tumor development (Yancey et al. Transglutaminase and tumorgrowth, Annals of the New York Academy of Sciences. 202. 344-8, 1972),and has been proposed to be a potential marker of apoptosis duringhormonal therapy and progression of prostate cancer (Rittmaster et al.(1999) J. Urology. 162: 2165-2169 and Pasquali et al. (1999) J. Clin.Endocrin. & Metabol. 84: 1463-1469).

[0288] KIAA 18 exhibited an expression pattern very similar to PSA andwas significantly up-regulated in LNCaP cancer cells upon treatment ofandrogen. KIAA 18 expression was also investigated in multiple prostatecancer tissue specimens, and the results demonstrated that the level ofKIAA 18 correlated with tumor stage. Therefore, KIAA 18 can be used as anew marker for the progression of prostate cancer and a target for drugdevelopment.

[0289] In contrast, KIAA 96 was down-regulated by androgen in LNCaPprostate cancer cells. Based on protein homology analysis, KIAA 96 wasfound to be a putative serine and threonine kinase, and was highlyhomologous to SNF1-related proteins, in particular, to mouse and ratSNF1-ratelated proteins. Kinases play important roles in cell cycle andproliferation. Solid tumor tissue analysis revealed that KIAA 96 levelsdecreased with tumor grade. It may be possible that dysregulation ofKIAA 96 could directly be involved in tumorgenesisand could thereforepotentially serve as a marker or target for anti-cancer drugdevelopment.

[0290] In summary, the KIAA candidate ARGs may be useful forunderstanding the molecular mechanisms leading to the proliferation,differentiation, and function of the normal and diseased human prostate.Collectively, these results demonstrate that KIAA 18 and KIAA 96 can beused as diagnostic markers and is important for prostate tumor growth.The involvement of KIAA 18 and KIAA 96 in prostate cancer asdemonstrated herein, and modifying the expression of KIAA 18 and or KIAA96 (up-regulated or downregulated) may provide a therapeutic effect indeterring the progression of prostate cancer. This modification may beby either existing agents, or novel agents identified by the screeningmethods of the invention.

Example 2 Screening for Compounds Useful for the Treatment of ProstateCancer

[0291] The cDNA and protein sequences KIAA 18 or KIAA 96 are availablein the public database Genbank with accession numbers, D13643 andD43636, respectively. The publications and sequence databases providethose skilled in the art with the genes needed to prepare thetransfected cell lines useful in for the following screening assays.

[0292] Test compounds potentially useful for the treatment of prostatecancer can be identified by expressing KIAA 18 or KIAA 96 in prostatecancer cells (e.g., LNCaP cells) which are stably transfected with avector capable of expressing KIAA 18 or KIAA 96 in the presence oftetraceycline (Tet-on system, available from Clontech). The transfectedLNCaP cells can be cultured under suitable conditions (e.g., in T175culture flasks in RPMI-1640 medium supplemented with 10%; fetal calfserum (FCS), 3 mM L-glutamine, 100 μg/ml streptomycin, and 100 units/mlpenicillin. To examine the effects of steroids, cells can be culturedfor 2 days in RPMI 1640 medium containing 5% FCS pretreated withdextrancoated charcoal (CT-FCS). The cells can be incubated in thepresence of a test compound with or without Tetracycline and the growthrate of the cells can be measured. A compound that demonstratesdifferential inhibitory activity in cells treated with Tet versus thosenot treated with Tet is a potential therapeutic compound for thetreatment of prostate cancer.

[0293] To find specific inhibitors of KIAA 96, a high throughput assaywill be established to screen a library of compounds. The expressed KIAA96 protein will be isolated from cells using standard isolationtechniques, and the isolated protein will be tested using atime-resolved fluorometric kinase assay. Suitable substrates for use inthe kinase assay include general substrates such as, histone or casein.Briefly, KIAA 96 and GST (Glutathione transferase)-fused substrate(e.g., histone or casein) can be incubated at 30° C. for 15 min in thepresence of ATP to allow phosphorylation of the substrate by KIAA 96.The GST-substrate can be captured by anti-GST antibody coated onto a 96well plate. Phosphorylated substrate can be detected using a primaryantibody recognizing only the phosphorylated serine or threonineresidues of the GST-substrate. Europium (Eu)-labeled secondary antibodycan be added to the 96 well plate to detect the primary antibody. Theplate can then incubated in enhancement buffer for 30 min beforedetection with a multilabel counter (1420 VICTOR², EG&G Wallac, Inc.).Compounds that alter the phosphate activity of KIAA 96 can be furtherevaluated using cell based assays to determine their effect on thegrowth rate of prostate cancer cells.

[0294] To evaluate the role of KIAA 18, LNCaP cells can be transfectedwith an expression vector containing the KIAA 18 cDNA (accession numberD13643), or with a control vector without the KIAA 18 cDNA insert. Aftertransfection and expression of KIAA 18, the cells can be monitored forthe effect of KIAA 18 on cell growth rate. A change in cell growth ratedemonstrates the role of KIAA 18 in the regulation of tumor cell growth.

[0295] As an alternative method to monitor the effects of KIAA 18, theKIAA 18 protein can be delivered directly to LNCaP cells by linking theKIAA18 protein to a cell-penetrating peptide such as TAT fragment. For acontrol experiment, the cells can be cultured in the presence of the TATfragment alone. Cells can be cultured in the presence of the linked KIAA18-TAT fragment for a time period that allows the KIAA 18-TAT fragmentto be taken up by the cells. Cell growth can be monitored and theeffects of KIAA 18 on cell growth can be determined.

Example 3 Detection of KIAA Markers

[0296] To evaluate the role of KIAA markers, e.g., KIAA 18 and/or KIAA96 in cell growth and the effect in tumor inhibition, the growth rate ofcells transfected with, for example, a KIAA 18 expression vector, orempty vector, will be determined. Altered growth will confirm the roleof the transglutaminase, KIAA 18 in the regulation of tumor cell growthand assure the therapeutical value of transglutaminase. The presence andexpression levels of the KIAA markers can be assessed using standardmolecular biology techniques as described in Sambrook et al, (1989)supra.

[0297] For the detection and quantitation of RNA species, the nucleicacids corresponding to the KIAA markers can be isolated and amplified.Pairs of primers that selectively hybridize to KIAA 18 and KIAA 96nucleic acid can be designed based on the nucleotide sequences of thesemarkers, which are available from Genbank accession numbers, D13643 andD43636, respectively. The primers can be contacted with the isolatednucleic acid under conditions that allow selective hybridization. Oncehybridized, the nucleic acid:primer complex can be contacted with one ormore enzymes that facilitate template-dependent nucleic acid synthesisusing PCR amplification. The amplified product can be detected, forexample by gel electrophoresis and visualization with ethidium bromideunder UV light. Alternatively, if the amplification products can beintegrally labeled with radio- or fluorometrically-labeled nucleotides,the amplification products can then be exposed to x-ray film orvisualized under the appropriate stimulating spectra, followingseparation.

[0298] Other methods for detecting the presence and expression levels ofthe KIAA markers include detecting the KIAA maker proteins by an ELISAimmunodetection assay. For example, by using anti-KIAA 18 or anti-KIAA96 antibodies to detect the presence of the KIAA markers expressed in acell sample. Anti-KIAA antibodies can be immobilized onto a selectedsurface exhibiting protein affinity, such as a well in a polystyrenemicrotiter plate. Then, a cell sample suspected of containing the KIAAmarkers, can be added to the wells. After binding and washing to removenon-specifically bound immunocomplexes, the bound antibody may bedetected. Detection can be achieved by the addition of a second antibodyspecific for a different region of the KIAA 18 and KIAA 96 markerproteins, that is linked to a detectable label.

Example 4 Detection of KIAA Markers in Solid Tumors

[0299] To determine whether the KIAA genes were effected at differentstages of tumor growth,. Solid tumors were scored using the Gleasonscoring system (See e.g., Bostwick (1994) Amer. J. Clin. Path. 102:S38-56, incorporated herein by reference). RNA was isolated from normalprostate glands and prostate tumors with different Gleason grades of1,2, 5, 6 and 7, 8 as shown in Table 1. The total RNA was examined forthe level of expression of KIAA 18 and KIAA 96 in these differenttumors. TABLE 1 Differential expression of KIAA 18 and KIAA 96 withincrease in tumor grade Tumor Grade KIAA 18 KIAA 96 1, 2 1.6  −1.38 5, 63.26 −3.00 7, 8 2.68 −3.25

[0300] The results are represented as fold increase or decrease abovethe base line.

[0301] These results demonstrate that the level of expression of KIAA 18increase with an increase in tumor grade, particularly at a tumor gradeof 5 and 6 compared to a tumor grade of 1 and 2. In contrast, the levelof KIAA 96 expression decreases with an increase in tumor grade. Theseresults demonstrate that KIAA 18 and KIAA 96 may be used as markers tomonitor the progression of solid tumor growth.

[0302] Equivalents

[0303] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed:
 1. A method of assessing whether a subject is afflictedprostate cancer, the method comprising comparing: a) the level ofexpression of a marker in a sample from a subject, wherein the marker isselected from the group consisting of one or more KIAA markers, and b)the normal level of expression of the marker in a control sample,wherein a significant difference between the level of expression of themarker in the sample from the subject and the normal level is anindication that the subject is afflicted with prostate cancer.
 2. Themethod of claim 1, wherein the marker corresponds to a transcribedpolynucleotide or portion thereof, wherein the polynucleotide comprisesthe marker.
 3. The method of claim 1, wherein the sample comprises cellsobtained from the subject.
 4. The method of claim 3, wherein the cellsare collected from the prostate gland.
 5. The method of claim 3, whereinthe cells are collected from blood.
 6. The method of claim 1, whereinthe level of expression of the marker in the sample differs from thenormal level of expression of the marker in a subject not afflicted withprostate cancer by a factor of at least about
 2. 7. The method of claim1, wherein the level of expression of the marker in the sample differsfrom the normal level of expression of the marker in a subject notafflicted with prostate cancer by a factor of at least about
 3. 8. Themethod of claim 1, wherein the marker is not significantly expressed innon-prostate cancer cells.
 9. The method of claim 1, wherein the levelof expression of the marker in the sample is assessed by detecting thepresence in the sample of a protein corresponding to the marker.
 10. Themethod of claim 9, wherein the presence of the protein is detected usinga reagent which specifically binds with the protein.
 11. The method ofclaim 10, wherein the reagent is selected from the group consisting ofan antibody, an antibody derivative, and an antibody fragment.
 12. Themethod of claim 1, wherein the level of expression of the marker in thesample is assessed by detecting the presence in the sample of atranscribed polynucleotide or portion thereof, wherein the transcribedpolynucleotide comprises the marker.
 13. The method of claim 12, whereinthe transcribed polynucleotide is an mRNA.
 14. The method of claim 12,wherein the transcribed polynucleotide is a cDNA.
 15. The method ofclaim 12, wherein the step of detecting further comprises amplifying thetranscribed polynucleotide.
 16. The method of claim 1, wherein the levelof expression of the marker in the sample is assessed by detecting thepresence in the sample of a transcribed polynucleotide which annealswith the marker or anneals with a portion of a polynucleotide, whereinthe polynucleotide comprises the marker, under stringent hybridizationconditions.
 17. The method of claim 1, further comprising comparing: a)the level of expression in the sample of each of at least two KIAAmarkers independently, and b) the normal level of expression of at leasttwo KIAA markers in samples of the same type obtained from controlsubjects not afflicted prostate cancer, wherein the level of expressionof more than one of the markers is significantly altered, relative tothe corresponding normal levels of expression of the markers, is anindication that the subject is afflicted prostate cancer.
 18. A methodfor monitoring the progression of prostate cancer in a subject, themethod comprising: a) detecting in a subject sample at a first point intime, the expression of a marker, wherein the marker is selected fromthe group consisting of the markers KIAA 18 and KIAA 96 or a combinationthereof; b) repeating step a) at a subsequent point in time; and c)comparing the level of expression detected in steps a) and b), andtherefrom monitoring the progression of prostate cancer in the subject.19. The method of claim 18, wherein marker corresponds to a transcribedpolynucleotide or portion thereof, wherein the polynucleotide comprisesthe marker.
 20. The method of claim 18, wherein the sample comprisescells obtained from the subject.
 21. The method of claim 20, wherein thecells are collected from the prostate gland.
 22. The method of claim 20,wherein the cells are collected from blood.
 23. A method of assessingthe efficacy of a therapy for inhibiting prostate cancer in a subject,the method comprising comparing: a) expression of a KIAA 18 marker inthe first sample obtained from the subject prior to providing at least aportion of the therapy to the subject, and b) expression of the KIAA 18marker in a second sample obtained from the subject following provisionof the portion of the therapy, wherein a significantly lower level ofexpression of the marker in the second sample, relative to the firstsample, is an indication that the therapy is efficacious for inhibitingprostate cancer in the subject.
 24. A method of assessing the efficacyof a therapy for inhibiting prostate cancer in a subject, the methodcomprising comparing: a) expression of a KIAA 96 marker in the firstsample obtained from the subject prior to providing at least a portionof the therapy to the subject, and b) expression of the KIAA 96 markerin a second sample obtained from the subject following provision of theportion of the therapy, wherein a significantly enhanced level ofexpression of the marker in the second sample, relative to the firstsample, is an indication that the therapy is efficacious for inhibitingprostate cancer in the subject.
 25. A method of assessing the potentialof a test compound to trigger prostate cancer in a cell, the methodcomprising: a) maintaining separate aliquots of cells in the presenceand absence of the test compound; and b) comparing expression of a KIAA18 marker in each of the aliquots, wherein a significantly enhancedlevel of expression of the KIAA 18 marker in the aliquot maintained inthe presence of the test compound, relative to the aliquot maintained inthe absence of the test compound, is an indication that the testcompound possesses the potential for triggering prostate cancer in acell.
 26. A method of assessing the potential of a test compound totrigger prostate cancer in a cell, the method comprising: a) maintainingseparate aliquots of cells in the presence and absence of the testcompound; and b) comparing expression of a KIAA 96 marker in each of thealiquots, wherein a significantly reduced level of expression of theKIAA 96 marker in the aliquot maintained in the presence of the testcompound, relative to the aliquot maintained in the absence of the testcompound, is an indication that the test compound possesses thepotential for triggering prostate cancer in a cell.
 27. A method oftreating a subject afflicted with prostate cancer, the method comprisingproviding to cells of the subject an antisense oligonucleotidecomplementary to a polynucleotide corresponding to a KIAA 18 marker. 28.A method of inhibiting prostate cancer in a subject at risk fordeveloping prostate cancer, the method comprising inhibiting expressionof a gene corresponding to a KIAA 18 marker.
 29. A method of treating asubject afflicted with prostate cancer, the method comprisingadministering anti-KIAA 18 antibodies.
 30. A method of treating asubject afflicted with prostate cancer, the method comprising deliveringserine threonine kinase encoded by KIAA 96 to cells of the subject. 31.A method of treating a subject afflicted with prostate cancer, themethod comprising expressing KIAA 96 in the cells of the subject.
 32. Amethod for identifying a compound useful for treating prostate cancer,comprising: a) measuring the expression level of a KIAA 18 marker in acell in the presence of a test compound; and b) comparing the expressionmeasured in step a) to the expression of the KIAA 18 marker in a cell inthe absence of the compound, wherein the compound is useful for treatingprostate cancer when the expression level of the KIAA 18 marker in thepresence of the test compound is lower than its expression level in theabsence of the test compound.
 33. The method of claim 32, wherein theexpression level is determined by measuring the levels of mRNA of theKIAA 18 marker.
 34. The method of claim 32, wherein the expression levelis determined by measuring the levels of the protein of the KIAA 18marker.
 35. The method of claim 32, wherein the cell is a prostatecancer cell.
 36. A method for identifying a compound useful for treatingprostate cancer, comprising: a) measuring the expression level of a KIAA96 marker in a cell in the presence of a test compound; and b) comparingthe expression measured in step a) to the expression of the KIAA 96marker in a cell in the absence of the compound, wherein the compound isuseful for treating prostate cancer when the expression level of theKIAA 96 marker in the presence of the test compound is higher than itsexpression level in the absence of the test compound.
 37. The method ofclaim 36, wherein the expression level is determined by measuring thelevels of mRNA of the KIAA 96 marker.
 38. The method of claim 36,wherein the expression level is determined by measuring the levels ofthe protein of the KIAA 96 marker.
 39. The method of claim 36, whereinthe cell is a prostate cancer cell.
 40. A method for identifying acompound useful for treating prostate cancer, comprising a) measuring anactivity of a KIAA 18 marker; and b) comparing the activity measured instep a) to the level of activity of the KIAA 18 marker in the absence ofthe test compound, wherein the compound is useful for treating prostatecancer when the activity of the KIAA 18 marker in the presence of thetest compound is lower than its activity in the absence of the testcompound.
 41. The method of claim 40, wherein the cell is a prostatecancer cell.
 42. A method for identifying a compound useful for treatingprostate cancer, comprising a) measuring an activity of a markerselected from a KIAA 96 marker; and b) comparing the activity measuredin step a) to the level of activity of the KIAA 96 marker in the absenceof the test compound, wherein the compound is useful for treatingprostate cancer when the activity of the KIAA 96 marker in the presenceof the test compound is higher than its activity in the absence of thetest compound.
 43. A method of treating prostate cancer in a patient,comprising administering to the patient a compound which decreases theexpression of a KIAA 18 marker.
 44. The method of claim 43, wherein thecompound decreases expression of mRNA of the KIAA 18 marker.
 45. Themethod of claim 43, wherein the compound decreases expression of theKIAA 18 marker protein.
 46. A method of treating prostate cancer in apatient, comprising administering to the patient a compound whichincreases the expression of a KIAA 96 marker.
 47. The method of claim46, wherein the compound increases expression of mRNA of the KIAA 96marker.
 48. The method of claim 46, wherein the compound increasesexpression of the KIAA 96 marker protein.
 49. A method for determiningthe efficacy of androgen withdrawal treatment in a subject afflictedwith prostate cancer, comprising: a) detecting in a subject sample at afirst point in time, the expression level of a marker, wherein themarker is selected from the group consisting of KIAA 18; b) repeatingstep a) at a subsequent point in time occurring after the subject beginsandrogen withdrawal treatment; and c) comparing the level of expressionof markers detected in steps a) and b), wherein a decrease in the levelof expression indicates that the androgen withdrawal treatment hasreduced efficacy.
 50. A method for determining the efficacy of androgenwithdrawal treatment in a subject afflicted with prostate cancer,comprising: a) detecting in a subject sample at a first point in time,the expression level of a marker, wherein the marker is selected fromthe group consisting of KIAA 96; b) repeating step a) at a subsequentpoint in time occurring after the subject begins androgen withdrawaltreatment; and c) comparing the level of expression of markers detectedin steps a) and b), wherein an increase in the level of expressionindicates that the androgen withdrawal treatment has reduced efficacy.